Anti-Jagged 1/Jagged 2 Cross-Reactive Antibodies, Activatable Anti-Jagged Antibodies And Methods Of Use Thereof

ABSTRACT

This invention relates generally to the generation of antibodies, e.g., monoclonal antibodies including fully human monoclonal antibodies, that recognize Jagged 1 and/or Jagged 2, to antibodies, e.g., monoclonal antibodies including fully human antibodies that recognize Jagged 1 and/or Jagged 2, and nucleic acid molecules that encode antibodies, e.g., nucleic acid molecules that encode monoclonal antibodies including fully human cross-reactive antibodies that recognize both Jagged 1 and Jagged 2, and to methods of making the anti-Jagged antibodies and methods of using the anti-Jagged antibodies as therapeutics, prophylactics, and diagnostics. The invention also relates generally to activatable antibodies that include a masking moiety (MM), a cleavable moiety (CM), and an antibody (AB) that specifically bind to Jagged 1 and Jagged 2, and to methods of making and using these activatable anti-Jagged antibodies in a variety of therapeutic, diagnostic and prophylactic indications.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/663,307, filed Jun. 22, 2012; U.S. Provisional Application No.61/749,212, filed Jan. 4, 2013; U.S. Provisional Application No.61/749,486, filed Jan. 7, 2013; and U.S. Provisional Application No.61/755,810, filed Jan. 23, 2013; each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to the generation of antibodies, e.g.,monoclonal antibodies including fully human monoclonal antibodies, thatrecognize Jagged 1 and/or Jagged 2, to antibodies, e.g., monoclonalantibodies including fully human antibodies that recognize Jagged 1and/or Jagged 2, and nucleic acid molecules that encode antibodies,e.g., nucleic acid molecules that encode monoclonal antibodies includingfully human cross-reactive antibodies that recognize both Jagged 1 andJagged 2, and to methods of making the anti-Jagged antibodies andmethods of using the anti-Jagged antibodies as therapeutics,prophylactics, and diagnostics. The invention also relates generally toactivatable antibodies that specifically bind to Jagged 1 and Jagged 2,and methods of making and using these activatable anti-Jagged antibodiesin a variety of therapeutic, diagnostic and prophylactic indications.

BACKGROUND OF THE INVENTION

The Notch signaling pathway regulates a wide variety of cell types andcellular processes. The Notch signaling pathway is regulated by ligandbinding, including ligands such as Jagged 1 and Jagged 2. Accordingly,there exists a need for therapeutics and diagnostics that target theNotch signaling pathway, including Jagged 1 and/or Jagged 2.

SUMMARY OF THE INVENTION

The present invention describes compositions for the diagnosis and/ortreatment of cancer or fibrotic disease. Specifically, this inventionprovides antibodies that bind both Jagged 1 and Jagged 2 and inhibittheir binding to and signaling through Notch receptors. The inventionprovides monoclonal antibodies that specifically bind to Jagged 1 andJagged 2. The antibodies of the invention modulate, e.g., block,inhibit, reduce, antagonize, neutralize or otherwise interfere withbinding of Jagged 1 to Notch receptors, binding of Jagged 2 to Notchreceptors, binding of Jagged 1 and Jagged 2 to Notch Receptors,signaling through the interaction between Jagged 1 and Notch receptors,signaling through the interaction between Jagged 2 and Notch receptors,and/or signaling through the interaction among both Jagged 1, Jagged 2and Notch receptors. These antibodies are referred to herein as“anti-Jagged antibodies.” The anti-Jagged antibodies of the inventioninclude monoclonal antibodies, such as, for example, fully humanmonoclonal antibodies, as well as humanized monoclonal antibodies andchimeric antibodies. In some embodiments, the antibodies are IgGisotype. In some embodiments, the antibodies are IgG1 isotype. In someembodiments, the antibodies have one of any of the isotypes disclosedherein.

Jagged 1, initially identified as Jagged and also referred to as JAG1,JAGL1 and/or HJ1, has been shown to be a transmembrane ligand for Notch.Jagged 2 is another ligand of Notch that is related to Jagged 1. BothJagged 1 and Jagged 2 are ligands for Notch-1, Notch-2, Notch-3 andNotch-4 receptors. (See e.g., Shimizu K et al., “Binding of Delta1,Jagged 1, and Jagged 2 to Notch2 rapidly induces cleavage, nucleartranslocation, and hyperphosphorylation of Notch2.” Molecular CellularBiology 20(18): 6913-6922 (2000); Shimizu K et al., “Physicalinteraction of Delta1, Jagged 1, and Jagged 2 with Notch1 and Notch3receptors. Biochemical and Biophysical Research Communications 276(1):385-389 (2000); Microvasc Res. 60(2):91-103 (2000)). Jagged proteinswere originally referred to as Serrate proteins.

Exemplary monoclonal antibodies of the invention include, for example,the 4D11 antibody, the 4B2 antibody, the 4E7 antibody, the 4E11antibody, the 6B7 antibody, and the 6F8 antibody. Other suitableantibodies include an antibody that binds to the same epitope as the4D11 antibody, the 4B2 antibody, the 4E7 antibody, the 4E11 antibody,the 6B7 antibody, and/or the 6F8 antibody.

These antibodies show specificity for human Jagged 1 and human Jagged 2,and they have been shown to inhibit or otherwise interfere with Jagged 1and/or Jagged 2 mediated signaling through Notch receptors. Theseantibodies also show specificity for mouse and rat Jagged 1. In someembodiments, these antibodies also show specificity for mouse and/or ratJagged 2.

In some embodiments, anti-Jagged antibodies of the disclosure can beinternalized upon binding to Jagged 1 and/or Jagged 2 expressed on adiseased cell, e.g., on a cancer cell or a cell involved in a fibroticdisorder.

The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VHCDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence selected from the combinations shown in Table 2. Theanti-Jagged antibodies of the invention include antibodies that containa combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequencethat are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the sequences shown in Table 2.

The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VHCDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence of at least one antibody selected from the group consisting ofthe 4D11 antibody, the 4B2 antibody, the 4E7 antibody, the 4E11antibody, the 6B7 antibody, and the 6F8 antibody. The anti-Jaggedantibodies of the invention include antibodies that contain acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequencethat are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the respective CDR sequences of at least one antibodyselected from the group consisting of the 4D11 antibody, the 4B2antibody, the 4E7 antibody, the 4E11 antibody, the 6B7 antibody, and the6F8 antibody.

The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VHCDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein at least one CDR sequence is selected from the groupconsisting of a VH CDR1 sequence that includes at least the amino acidsequence SYAMS (SEQ ID NO: 200); a VH CD2 sequence that includes atleast the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VHCDR3 sequence that includes at least the amino acid sequence DIGGRSAFDY(SEQ ID NO: 209); a VL CDR1 sequence that includes at least the aminoacid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes at least the amino acid sequence AASSLQS (SEQ ID NO: 211); anda VL CDR3 sequence that includes at least the amino acid sequenceQQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VHCDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein at least one CDR sequence is selected from the groupconsisting of a VH CDR1 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence SYAMS (SEQ ID NO: 200); a VH CD2 sequencethat includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequenceSIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VH CDR3 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO:209); a VL CDR1 sequence that includes a sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQID NO: 211); and a VL CDR3 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence QQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a VH CDR1 sequence that includes at least the amino acidsequence SYAMS (SEQ ID NO: 200); a VH CD2 sequence that includes atleast the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VHCDR3 sequence that includes at least the amino acid sequence DIGGRSAFDY(SEQ ID NO: 209); a VL CDR1 sequence that includes at least the aminoacid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes at least the amino acid sequence AASSLQS (SEQ ID NO: 211); anda VL CDR3 sequence that includes at least the amino acid sequenceQQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a VH CDR1 sequence that includes a sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe amino acid sequence SYAMS (SEQ ID NO: 200); a VH CD2 sequence thatincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequenceSIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VH CDR3 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO:209); a VL CDR1 sequence that includes a sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQID NO: 211); and a VL CDR3 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence QQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a variable heavy chain region and a variablelight chain region selected from the combinations listed in Table 4. Theanti-Jagged antibodies of the invention include antibodies that containa combination of a variable heavy chain region and a variable lightchain region that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identical to the combinations listed in Table 4.

In some embodiments, the anti-Jagged antibody includes a light chainsequence that includes SEQ ID NO: 74. In some embodiments, theanti-Jagged antibody includes a light chain sequence that includes anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:74. In some embodiments, the anti-Jagged antibody includes a heavy chainsequence that includes SEQ ID NO: 76. In some embodiments, theanti-Jagged antibody includes a heavy chain sequence that includes anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:76. In some embodiments, the anti-Jagged antibody includes a light chainsequence that includes SEQ ID NO: 74 a heavy chain sequence thatincludes SEQ ID NO: 76. In some embodiments, the anti-Jagged antibodyincludes a light chain sequence that includes an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 74 and a heavychain sequence that includes an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe amino acid sequence of SEQ ID NO: 76.

In some embodiments, the anti-Jagged antibody includes a light chainsequence that includes SEQ ID NO: 54. In some embodiments, theanti-Jagged antibody includes a light chain sequence that includes anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:54. In some embodiments, the anti-Jagged antibody includes a heavy chainsequence that includes SEQ ID NO: 56. In some embodiments, theanti-Jagged antibody includes a heavy chain sequence that includes anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:56. In some embodiments, the anti-Jagged antibody includes a light chainsequence that includes SEQ ID NO: 54 a heavy chain sequence thatincludes SEQ ID NO: 56. In some embodiments, the anti-Jagged antibodyincludes a light chain sequence that includes an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 54 and a heavychain sequence that includes an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe amino acid sequence of SEQ ID NO: 56.

In some embodiments, the anti-Jagged antibody also includes an agentconjugated to the AB. In some embodiments, the agent is a therapeuticagent. In some embodiments, the agent is an antineoplastic agent. Insome embodiments, the agent is a toxin or fragment thereof. In someembodiments, the agent is conjugated to the anti-Jagged antibody via alinker. In some embodiments, the linker is a cleavable linker. In someembodiments, the agent is an agent selected from the group listed inTable 30. In some embodiments, the agent is a dolastatin. In someembodiments, the agent is an auristatin or derivative thereof. In someembodiments, the agent is auristatin E or a derivative thereof. In someembodiments, the agent is monomethyl auristatin E (MMAE). In someembodiments, the agent is a maytansinoid or maytansinoid derivative. Insome embodiments, the agent is DM1 or DM4. In some embodiments, theagent is a duocarmycin or derivative thereof. In some embodiments, theagent is a calicheamicin or derivative thereof.

In some embodiments, the anti-Jagged antibody also includes a detectablemoiety. In some embodiments, the detectable moiety is a diagnosticagent.

In some embodiments, the anti-Jagged antibody naturally contains one ormore disulfide bonds. In some embodiments, the anti-Jagged antibody canbe engineered to include one or more disulfide bonds.

The invention also provides an isolated nucleic acid molecule encodingan anti-Jagged antibody described herein, as well as vectors thatinclude these isolated nucleic acid sequences. The invention providesmethods of producing an anti-Jagged antibody by culturing a cell underconditions that lead to expression of the anti-Jagged antibody, whereinthe cell comprises such a nucleic acid molecule. In some embodiments,the cell comprises such a vector.

The invention also provides activatable antibodies and activatableantibody compositions that include an antibody or antigen-bindingfragment thereof that specifically binds Jagged 1 and Jagged 2 coupledor otherwise attached to a masking moiety (MM), such that coupling ofthe MM reduces the ability of the antibody or antigen-binding fragmentthereof to bind Jagged 1 and Jagged 2. These activatable antibodies arecollectively referred to herein as activatable anti-Jagged antibodies,also referred to herein as anti-Jagged activatable antibodies or Jaggedactivatable antibodies. In some embodiments, the MM is coupled via asequence that includes a substrate for a protease, for example, aprotease that is co-localized with Jagged 1 and/or Jagged 2 at atreatment site or a diagnostic site in a subject. The activatableanti-Jagged antibodies provided herein are stable in circulation,activated at intended sites of therapy and/or diagnosis but not innormal, i.e., healthy tissue, and, when activated, exhibit binding toJagged 1 and Jagged 2 that is at least comparable to the corresponding,unmodified antibody.

The activatable anti-Jagged antibodies of the invention includeantibodies that contain a combination of a VH CDR1 sequence, a VH CDR2sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence,and a VL CDR3 sequence selected from the combinations shown in Table 2.The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VHCDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the sequences shown in Table 2.

The activatable anti-Jagged antibodies of the invention includeantibodies that contain a combination of a VH CDR1 sequence, a VH CDR2sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence,and a VL CDR3 sequence of at least one antibody selected from the groupconsisting of the 4D11 antibody, the 4B2 antibody, the 4E7 antibody, the4E11 antibody, the 6B7 antibody, and the 6F8 antibody. The anti-Jaggedantibodies of the invention include antibodies that contain acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequencethat are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the respective CDR sequences of at least one antibodyselected from the group consisting of the 4D11 antibody, the 4B2antibody, the 4E7 antibody, the 4E11 antibody, the 6B7 antibody, and the6F8 antibody.

The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VHCDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein at least one CDR sequence is selected from the groupconsisting of a VH CDR1 sequence that includes at least the amino acidsequence SYAMS (SEQ ID NO: 200); a VH CD2 sequence that includes atleast the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VHCDR3 sequence that includes at least the amino acid sequence DIGGRSAFDY(SEQ ID NO: 209); a VL CDR1 sequence that includes at least the aminoacid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes at least the amino acid sequence AASSLQS (SEQ ID NO: 211); anda VL CDR3 sequence that includes at least the amino acid sequenceQQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VHCDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein at least one CDR sequence is selected from the groupconsisting of a VH CDR1 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence SYAMS (SEQ ID NO: 200); a VH CD2 sequencethat includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequenceSIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VH CDR3 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO:209); a VL CDR1 sequence that includes a sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQID NO: 211); and a VL CDR3 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence QQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a VH CDR1 sequence that includes at least the amino acidsequence SYAMS (SEQ ID NO: 200); a VH CD2 sequence that includes atleast the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VHCDR3 sequence that includes at least the amino acid sequence DIGGRSAFDY(SEQ ID NO: 209); a VL CDR1 sequence that includes at least the aminoacid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes at least the amino acid sequence AASSLQS (SEQ ID NO: 211); anda VL CDR3 sequence that includes at least the amino acid sequenceQQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a VH CDR1 sequence that includes a sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe amino acid sequence SYAMS (SEQ ID NO: 200); a VH CD2 sequence thatincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequenceSIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VH CDR3 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO:209); a VL CDR1 sequence that includes a sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQID NO: 211); and a VL CDR3 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence QQTVVAPPL (SEQ ID NO: 212).

The activatable anti-Jagged antibodies of the invention includeantibodies that contain a combination of a variable heavy chain regionand a variable light chain region selected from the combinations listedin Table 4. The anti-Jagged antibodies of the invention includeantibodies that contain a combination of a variable heavy chain regionand a variable light chain region that are at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the combinationslisted in Table 4.

In some embodiments, the anti-Jagged antibody of the activatableantibody includes a light chain sequence that includes SEQ ID NO: 74. Insome embodiments, the anti-Jagged antibody includes a light chainsequence that includes an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the aminoacid sequence of SEQ ID NO: 74. In some embodiments, the anti-Jaggedantibody includes a heavy chain sequence that includes SEQ ID NO: 76. Insome embodiments, the anti-Jagged antibody includes a heavy chainsequence that includes an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the aminoacid sequence of SEQ ID NO: 76. In some embodiments, the anti-Jaggedantibody includes a light chain sequence that includes SEQ ID NO: 74 anda heavy chain sequence that includes SEQ ID NO: 76. In some embodiments,the anti-Jagged antibody includes a light chain sequence that includesan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQID NO: 74 and a heavy chain sequence that includes an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence of SEQ ID NO: 76.

In some embodiments, the anti-Jagged antibody includes a light chainsequence that includes an amino acid sequence selected from the groupconsisting of SEQ ID NO: 74, 132, 134, 136, 138, 140, 142, 144 and 146.In some embodiments, the anti-Jagged antibody includes a light chainsequence that includes an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an aminoacid sequence selected from the group consisting of SEQ ID NO: 74, 132,134, 136, 138, 140, 142, 144 and 146. In some embodiments, theanti-Jagged antibody includes a heavy chain sequence that includes SEQID NO: 76 or SEQ ID NO: 148. In some embodiments, the anti-Jaggedantibody includes a heavy chain sequence that includes an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 74, 132, 134, 136, 138, 140, 142, 144 and 146.In some embodiments, the anti-Jagged antibody includes a light chainsequence that includes an amino acid sequence selected from the groupconsisting of SEQ ID NO: 74, 132, 134, 136, 138, 140, 142, 144 and 146and a heavy chain sequence that includes SEQ ID NO: 76 or SEQ ID NO:148. In some embodiments, the anti-Jagged antibody includes a lightchain sequence that includes an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to anamino acid sequence selected from the group consisting of SEQ ID NO: 74,132, 134, 136, 138, 140, 142, 144 and 146 and a heavy chain sequencethat includes an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence of SEQ ID NO: 76 or SEQ ID NO: 148.

The activatable antibodies described herein in an activated state bindJagged 1 and Jagged 2 and include (i) an antibody or an antigen bindingfragment thereof (AB) that specifically binds to Jagged 1 and Jagged 2;(ii) a masking moiety (MM) that inhibits the binding of the AB to Jagged1 and Jagged 2 when the activatable antibody is in an uncleaved state;and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is apolypeptide that functions as a substrate for a protease. In someembodiments, the MM is coupled to the AB via the CM.

In some embodiments, the activatable antibody has the structuralarrangement from N-terminus to C-terminus as follows in the uncleavedstate: MM-CM-AB or AB-CM-MM.

In some embodiments, the activatable antibody comprises a linkingpeptide between the MM and the CM.

In some embodiments, the activatable antibody comprises a linkingpeptide between the CM and the AB.

In some embodiments, the activatable antibody comprises a first linkingpeptide (LP1) and a second linking peptide (LP2), and wherein theactivatable antibody has the structural arrangement from N-terminus toC-terminus as follows in the uncleaved state: MM-LP1-CM-LP2-AB orAB-LP2-CM-LP1-MM. In some embodiments, the two linking peptides need notbe identical to each other.

In some embodiments, at least one of LP1 or LP2 includes an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 123) and (GGGS)_(n) (SEQ ID NO: 124), where n isan integer of at least one. In some embodiments, at least one of LP1 orLP2 includes an amino acid sequence selected from the group consistingof GGSG (SEQ ID NO: 125), GGSGG (SEQ ID NO: 126), GSGSG (SEQ ID NO:127), GSGGG (SEQ ID NO: 128), GGGSG (SEQ ID NO: 129), and GSSSG (SEQ IDNO: 130).

In some embodiments, the activatable antibody includes an antibody orantigen-binding fragment thereof that specifically binds Jagged 1 andJagged 2. In some embodiments, the antibody or immunologically activefragment thereof that binds Jagged 1 and Jagged 2 is a monoclonalantibody, domain antibody, single chain, Fab fragment, a F(ab′)₂fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody,or a single domain light chain antibody. In some embodiments, such anantibody or immunologically active fragment thereof that binds Jagged 1and Jagged 2 is a rodent (e.g., mouse or rat), chimeric, humanized orfully human monoclonal antibody.

In some embodiments, activated anti-Jagged antibodies of the disclosure(i.e., in a cleaved state) can be internalized upon binding to Jagged 1and/or Jagged 2 expressed on a diseased cell, e.g., on a cancer cell ora cell involved in a fibrotic disorder.

In some embodiments, the AB has an equilibrium dissociation constant ofabout 100 nM or less for binding to Jagged 1 and Jagged 2.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to Jagged 1 and Jagged 2.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is no more than the equilibrium dissociationconstant of the AB to Jagged 1 and Jagged 2.

In some embodiments, the MM does not interfere or compete with the ABfor binding to Jagged 1 and Jagged 2 when the activatable antibody is ina cleaved state.

In some embodiments, the MM is a polypeptide of about 2 to 40 aminoacids in length. In some embodiments, the MM is a polypeptide of no morethan 40 amino acids in length.

In some embodiments, the MM polypeptide sequence is different from thatof Jagged 1 and Jagged 2 and wherein the MM polypeptide sequence is nomore than 50% identical to any natural binding partner of the AB. Insome embodiments, the MM polypeptide sequence is different from that ofJagged 1 and Jagged 2 and wherein the MM polypeptide sequence is no morethan 25% identical to any natural binding partner of the AB. In someembodiments, the MM polypeptide sequence is different from that ofJagged 1 and Jagged 2 and wherein the MM polypeptide sequence is no morethan 10% identical to any natural binding partner of the AB.

In some embodiments, the coupling of the MM reduces the ability of theAB to bind Jagged 1 and Jagged 2 such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards Jagged 1 and Jagged 2is at least 20 times greater than the K_(d) of the AB when not coupledto the MM towards Jagged 1 and Jagged 2.

In some embodiments, the coupling of the MM reduces the ability of theAB to bind Jagged 1 and Jagged 2 such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards Jagged 1 and Jagged 2is at least 40 times greater than the K_(d) of the AB when not coupledto the MM towards Jagged 1 and Jagged 2.

In some embodiments, the coupling of the MM reduces the ability of theAB to bind Jagged 1 and Jagged 2 such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards Jagged 1 and Jagged 2is at least 100 times greater than the K_(d) of the AB when not coupledto the MM towards Jagged 1 and Jagged 2.

In some embodiments, the coupling of the MM reduces the ability of theAB to bind Jagged 1 and Jagged 2 such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards Jagged 1 and Jagged 2is at least 1000 times greater than the K_(d) of the AB when not coupledto the MM towards Jagged 1 and Jagged 2.

In some embodiments, the coupling of the MM reduces the ability of theAB to bind Jagged 1 and Jagged 2 such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards Jagged 1 and Jagged 2is at least 10,000 times greater than the K_(d) of the AB when notcoupled to the MM towards Jagged 1 and Jagged 2.

In some embodiments, in the presence of Jagged 1 and Jagged 2, the MMreduces the ability of the AB to bind Jagged 1 and Jagged 2 by at least90% when the CM is uncleaved, as compared to when the CM is cleaved whenassayed in vitro using a target displacement assay such as, for example,the assay described in PCT Publication Nos. WO 2009/025846 and WO2010/081173, the contents of each of which are hereby incorporated byreference in their entirety.

In some embodiments, the MM does not interfere or compete with the AB ofthe activatable antibody in a cleaved state for binding to the Jaggedtarget.

In some embodiments, the MM is an amino acid sequence selected from thegroup of those listed in Tables 9, 11-14, and 20-23.

In some embodiments, the protease is co-localized with Jagged 1 and/orJagged 2 in a tissue, and the protease cleaves the CM in the activatableantibody when the activatable antibody is exposed to the protease.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody toJagged 1 and Jagged 2 is reduced to occur with an equilibriumdissociation constant that is at least 20-fold greater than theequilibrium dissociation constant of an unmodified AB binding to Jagged1 and Jagged 2, and whereas in the cleaved state, the AB binds Jagged 1and Jagged 2.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody toJagged 1 and Jagged 2 is reduced to occur with an equilibriumdissociation constant that is at least 40-fold greater than theequilibrium dissociation constant of an unmodified AB binding to Jagged1 and Jagged 2, and whereas in the cleaved state, the AB binds Jagged 1and Jagged 2.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody toJagged 1 and Jagged 2 is reduced to occur with an equilibriumdissociation constant that is at least 50-fold greater than theequilibrium dissociation constant of an unmodified AB binding to Jagged1 and Jagged 2, and whereas in the cleaved state, the AB binds Jagged 1and Jagged 2.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody toJagged 1 and Jagged 2 is reduced to occur with an equilibriumdissociation constant that is at least 100-fold greater than theequilibrium dissociation constant of an unmodified AB binding to Jagged1 and Jagged 2, and whereas in the cleaved state, the AB binds Jagged 1and Jagged 2.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody toJagged 1 and Jagged 2 is reduced to occur with an equilibriumdissociation constant that is at least 200-fold greater than theequilibrium dissociation constant of an unmodified AB binding to Jagged1 and Jagged 2, and whereas in the cleaved state, the AB binds Jagged 1and Jagged 2.

In some embodiments, the CM is a polypeptide of up to 15 amino acids inlength.

In some embodiments, the CM includes the amino acid sequence LSGRSDNH(SEQ ID NO: 213). In some embodiments, the cleavable moiety is selectedfor use with a specific protease, for example a protease that is knownto be co-localized with the target of the activatable antibody. Forexample, suitable cleavable moieties for use in the activatableanti-Jagged antibodies of the disclosure are cleaved by at least aprotease such as urokinase, legumain, and/or MT-SP1 (matriptase) andinclude the sequence TGRGPSWV (SEQ ID NO: 214); SARGPSRW (SEQ ID NO:215); TARGPSFK (SEQ ID NO: 216); LSGRSDNH (SEQ ID NO: 213); GGWHTGRN(SEQ ID NO: 217); HTGRSGAL (SEQ ID NO: 218); PLTGRSGG (SEQ ID NO: 219);AARGPAIH (SEQ ID NO: 220); RGPAFNPM (SEQ ID NO: 221); SSRGPAYL (SEQ IDNO: 222); RGPATPIM (SEQ ID NO: 223); RGPA (SEQ ID NO: 224); GGQPSGMWGW(SEQ ID NO: 225); FPRPLGITGL (SEQ ID NO: 226); VHMPLGFLGP (SEQ ID NO:227); SPLTGRSG (SEQ ID NO: 228); SAGFSLPA (SEQ ID NO: 229); LAPLGLQRR(SEQ ID NO: 230); SGGPLGVR (SEQ ID NO: 231); and/or PLGL (SEQ ID NO:232).

In some embodiments, the CM is a substrate for a protease selected fromthe group consisting of those shown in Table 33. In some embodiments,the protease is selected from the group consisting of uPA, legumain,MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12, MMP-13, andMMP-14. In some embodiments, the protease is a cathepsin. In someembodiments, the CM is a substrate for a protease selected from thegroup consisting of uPA (urokinase plasminogen activator), legumain andMT-SP1 (matriptase). In some embodiments, the protease comprises uPA. Insome embodiments, the protease comprises legumain. In some embodiments,the protease comprises MT-SP1. In some embodiments, the proteasecomprises a matrix metalloproteinase (MMP).

In some embodiments, the CM is a substrate for at least two proteases.In some embodiments, each protease is selected from the group consistingof those shown in Table 33. In some embodiments, the CM is a substratefor at least two proteases, wherein one of the proteases is selectedfrom the group consisting of uPA, legumain and MT-SP1 and the otherprotease is selected from the group consisting of those shown in Table33. In some embodiments, the CM is a substrate for at least twoproteases selected from the group consisting of uPA, legumain andMT-SP1.

In some embodiments, the activatable antibody includes at least a firstCM and a second CM. In some embodiments, the first CM and the second CMare each polypeptides of no more than 15 amino acids long. In someembodiments, the first CM and the second CM in the activatable antibodyhave the structural arrangement from N-terminus to C-terminus as followsin the uncleaved state: MM-CM1-CM2-AB or AB-CM2-CM1-MM. In someembodiments, at least one of the first CM and the second CM is apolypeptide that functions as a substrate for a protease selected fromthe group consisting of uPA, legumain, and MT-SP1. In some embodiments,the first CM is cleaved by a first cleaving agent selected from thegroup consisting of uPA, legumain, and MT-SP1 in a target tissue and thesecond CM is cleaved by a second cleaving agent in a target tissue. Insome embodiments, the other protease is selected from the groupconsisting of those shown in Table 33. In some embodiments, the firstcleaving agent and the second cleaving agent are the same proteaseselected from the group consisting of uPA, legumain, and MT-SP1, and thefirst CM and the second CM are different substrates for the enzyme. Insome embodiments, the first cleaving agent and the second cleaving agentare the same protease selected from the group consisting of those shownin Table 33. In some embodiments, the first cleaving agent and thesecond cleaving agent are different proteases. In some embodiments, thefirst cleaving agent and the second cleaving agent are co-localized inthe target tissue. In some embodiments, the first CM and the second CMare cleaved by at least one cleaving agent in the target tissue.

In some embodiments, the activatable antibody is exposed to and cleavedby a protease such that, in the activated or cleaved state, theactivated antibody includes a light chain amino acid sequence thatincludes at least a portion of LP2 and/or CM sequence after the proteasehas cleaved the CM.

In some embodiments, the MM and the CM include an amino acid sequenceselected from the group consisting of SEQ ID NO: 182, 184, 186, 188,190, 192, 194, and 196.

In some embodiments, the activatable antibody also includes a signalpeptide. In some embodiments, the signal peptide is conjugated to theactivatable antibody via a spacer. In some embodiments, the spacer isconjugated to the activatable antibody in the absence of a signalpeptide. In some embodiments, the spacer is joined directly to the MM ofthe activatable antibody.

In some embodiments, the activatable antibody in an uncleaved statecomprises a spacer that is joined directly to the MM and has thestructural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB.In some embodiments, the spacer includes at least the amino acidsequence QGQSGQ (SEQ ID NO: 233). In some embodiments, the MM and spacerinclude the amino acid sequence of SEQ ID NO: 180. In some embodiments,the MM and spacer include the amino acid sequence QGQSGQCNIWLVGGDCRGWQG(SEQ ID NO: 234).

In some embodiments, the activatable antibody also includes an agentconjugated to the AB. In some embodiments, the agent is a therapeuticagent. In some embodiments, the agent is an antineoplastic agent. Insome embodiments, the agent is a toxin or fragment thereof. In someembodiments, the agent is conjugated to the AB via a linker In someembodiments, the linker is a cleavable linker. In some embodiments, theagent is an agent selected from the group listed in Table 30. In someembodiments, the agent is a dolastatin. In some embodiments, the agentis an auristatin or derivative thereof. In some embodiments, the agentis auristatin E or a derivative thereof. In some embodiments, the agentis monomethyl auristatin E (MMAE). In some embodiments, the agent is amaytansinoid or maytansinoid derivative. In some embodiments, the agentis DM1 or DM4. In some embodiments, the agent is a duocarmycin orderivative thereof. In some embodiments, the agent is a calicheamicin orderivative thereof.

In some embodiments, the activatable antibody also includes a detectablemoiety. In some embodiments, the detectable moiety is a diagnosticagent.

In some embodiments, the AB of the activatable antibody naturallycontains one or more disulfide bonds. In some embodiments, the AB can beengineered to include one or more disulfide bonds.

In some embodiments, the serum half-life of the activatable antibody isat least 5 days when administered to an organism. In some embodiments,the serum half-life of the activatable antibody is at least 4 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 3 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 2 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least24 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 20 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 18 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 16 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least14 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 12 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 10 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 8 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least6 hours when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 4 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 3 hours when administered to anorganism.

In some embodiments, the activatable anti-Jagged antibody and/orconjugated activatable anti-Jagged antibody is monospecific. In someembodiments, the activatable anti-Jagged antibody and/or conjugatedactivatable anti-Jagged antibody is multispecific, e.g., by way ofnon-limiting example, bispecific or trifunctional. In some embodiments,the activatable anti-Jagged antibody and/or conjugated activatableanti-Jagged antibody is formulated as part of a pro-Bispecific T CellEngager (BITE) molecule. In some embodiments, the activatableanti-Jagged antibody and/or conjugated activatable anti-Jagged antibodyis formulated as part of a pro-Chimeric Antigen Receptor (CAR) modifiedT cell or other engineered receptor.

The disclosure also provides compositions and methods that include anactivatable anti-Jagged antibody that includes an antibody or antibodyfragment (AB) that specifically binds a Jagged target (e.g., Jagged 1and/or Jagged 2), where the AB is coupled to a masking moiety (MM) thatdecreases the ability of the AB to bind its target. In some embodiments,the activatable anti-Jagged antibody further includes a cleavable moiety(CM) that is a substrate for a protease. The compositions and methodsprovided herein enable the attachment of one or more agents to one ormore cysteine residues in the AB without compromising the activity(e.g., the masking, activating or binding activity) of the activatableanti-Jagged antibody. In some embodiments, the compositions and methodsprovided herein enable the attachment of one or more agents to one ormore cysteine residues in the AB without reducing or otherwisedisturbing one or more disulfide bonds within the MM. The compositionsand methods provided herein produce an activatable anti-Jagged antibodythat is conjugated to one or more agents, e.g., any of a variety oftherapeutic, diagnostic and/or prophylactic agents, preferably withoutany of the agent(s) being conjugated to the MM of the activatableanti-Jagged antibody. The compositions and methods provided hereinproduce conjugated activatable anti-Jagged antibodies in which the MMretains the ability to effectively and efficiently mask the AB of theactivatable antibody in an uncleaved state. The compositions and methodsprovided herein produce conjugated activatable anti-Jagged antibodies inwhich the activatable antibody is still activated, i.e., cleaved, in thepresence of a protease that can cleave the CM.

The activatable anti-Jagged antibodies have at least one point ofconjugation for an agent, but in the methods and compositions providedherein less than all possible points of conjugation are available forconjugation to an agent. In some embodiments, the one or more points ofconjugation are sulfur atoms involved in disulfide bonds. In someembodiments, the one or more points of conjugation are sulfur atomsinvolved in interchain disulfide bonds. In some embodiments, the one ormore points of conjugation are sulfur atoms involved in interchainsulfide bonds, but not sulfur atoms involved in intrachain disulfidebonds. In some embodiments, the one or more points of conjugation aresulfur atoms of cysteine or other amino acid residues containing asulfur atom. Such residues may occur naturally in the antibody structureor may be incorporated into the antibody by site-directed mutagenesis,chemical conversion, or mis-incorporation of non-natural amino acids.

Also provided are methods of preparing a conjugate of an activatableanti-Jagged antibody having one or more interchain disulfide bonds inthe AB and one or more intrachain disulfide bonds in the MM, and a drugreactive with free thiols is provided. The method generally includespartially reducing interchain disulfide bonds in the activatableantibody with a reducing agent, such as, for example, TCEP; andconjugating the drug reactive with free thiols to the partially reducedactivatable antibody. As used herein, the term partial reduction refersto situations where an activatable anti-Jagged antibody is contactedwith a reducing agent and less than all disulfide bonds, e.g., less thanall possible sites of conjugation are reduced. In some embodiments, lessthan 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or less than 5% of allpossible sites of conjugation are reduced.

In yet other embodiments, a method of reducing and conjugating an agent,e.g., a drug, to an activatable anti-Jagged antibody resulting inselectivity in the placement of the agent is provided. The methodgenerally includes partially reducing the activatable anti-Jaggedantibody with a reducing agent such that any conjugation sites in themasking moiety or other non-AB portion of the activatable antibody arenot reduced, and conjugating the agent to interchain thiols in the AB.The conjugation site(s) are selected so as to allow desired placement ofan agent to allow conjugation to occur at a desired site. The reducingagent is, for example, TCEP. The reduction reaction conditions such as,for example, the ratio of reducing agent to activatable antibody, thelength of incubation, the temperature during the incubation, the pH ofthe reducing reaction solution, etc., are determined by identifying theconditions that produce a conjugated activatable antibody in which theMM retains the ability to effectively and efficiently mask the AB of theactivatable antibody in an uncleaved state. The ratio of reduction agentto activatable anti-Jagged antibody will vary depending on theactivatable antibody. In some embodiments, the ratio of reducing agentto activatable anti-Jagged antibody will be in a range from about 20:1to 1:1, from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to1:1, from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to1:1, from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about 9:1to 1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5, from about6:1 to 1:1.5, from about 5:1 to 1:1.5, from about 4:1 to 1:1.5, fromabout 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5,or from about 1:1 to 1:1.5. In some embodiments, the ratio is in a rangeof from about 5:1 to 1:1. In some embodiments, the ratio is in a rangeof from about 5:1 to 1.5:1. In some embodiments, the ratio is in a rangeof from about 4:1 to 1:1. In some embodiments, the ratio is in a rangefrom about 4:1 to 1.5:1.

In some embodiments, a method of reducing interchain disulfide bonds inthe AB of an activatable anti-Jagged antibody and conjugating an agent,e.g., a thiol-containing agent such as a drug, to the resultinginterchain thiols to selectively locate agent(s) on the AB is provided.The method generally includes partially reducing the AB with a reducingagent to form at least two interchain thiols without forming allpossible interchain thiols in the activatable antibody; and conjugatingthe agent to the interchain thiols of the partially reduced AB. Forexample, the AB of the activatable antibody is partially reduced forabout 1 hour at about 37° C. at a desired ratio of reducingagent:activatable antibody. In some embodiments, the ratio of reducingagent to activatable antibody will be in a range from about 20:1 to 1:1,from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to 1:1,from about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to 1:1,from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to 1:1,from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about 9:1 to1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5, from about 6:1to 1:1.5, from about 5:1 to 1:1.5, from about 4:1 to 1:1.5, from about3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5, orfrom about 1:1 to 1:1.5. In some embodiments, the ratio is in a range offrom about 5:1 to 1:1. In some embodiments, the ratio is in a range offrom about 5:1 to 1.5:1. In some embodiments, the ratio is in a range offrom about 4:1 to 1:1. In some embodiments, the ratio is in a range fromabout 4:1 to 1.5:1.

The thiol-containing reagent can be, for example, cysteine or N-acetylcysteine. The reducing agent can be, for example, TCEP. In someembodiments, the reduced activatable antibody can be purified prior toconjugation, using for example, column chromatography, dialysis, ordiafiltration. Alternatively, the reduced antibody is not purified afterpartial reduction and prior to conjugation.

The invention also provides partially reduced activatable anti-Jaggedantibodies in which at least one interchain disulfide bond in theactivatable antibody has been reduced with a reducing agent withoutdisturbing any intrachain disulfide bonds in the activatable antibody,wherein the activatable antibody includes an antibody or an antigenbinding fragment thereof (AB) that specifically binds to a Jagged target(e.g., Jagged 1 and/or Jagged 2), a masking moiety (MM) that inhibitsthe binding of the AB of the activatable antibody in an uncleaved stateto the Jagged target, and a cleavable moiety (CM) coupled to the AB,wherein the CM is a polypeptide that functions as a substrate for aprotease. In some embodiments the MM is coupled to the AB via the CM. Insome embodiments, one or more intrachain disulfide bond(s) of theactivatable antibody is not disturbed by the reducing agent. In someembodiments, one or more intrachain disulfide bond(s) of the MM withinthe activatable antibody is not disturbed by the reducing agent. In someembodiments, the activatable antibody in the uncleaved state has thestructural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP.

In some embodiments, the anti-Jagged antibodies and/or the activatableanti-Jagged antibodies described herein are used in conjunction with oneor more additional agents or a combination of additional agents.Suitable additional agents include current pharmaceutical and/orsurgical therapies for an intended application, such as, for example,cancer. For example, the anti-Jagged antibodies and/or activatableanti-Jagged antibodies can be used in conjunction with an additionalchemotherapeutic or anti-neoplastic agent. For example, in someembodiments, the additional chemotherapeutic agent is gemcitabine.

In some embodiments, the anti-Jagged antibody and/or activatableanti-Jagged antibody and additional agent are formulated into a singletherapeutic composition, and the anti-Jagged antibody and/or activatableanti-Jagged antibody and additional agent are administeredsimultaneously. Alternatively, the anti-Jagged antibody and/oractivatable anti-Jagged antibody and additional agent are separate fromeach other, e.g., each is formulated into a separate therapeuticcomposition, and the anti-Jagged antibody and/or activatable anti-Jaggedantibody and the additional agent are administered simultaneously, orthe anti-Jagged antibody and/or activatable anti-Jagged antibody and theadditional agent are administered at different times during a treatmentregimen. For example, the anti-Jagged antibody and/or activatableanti-Jagged antibody is administered prior to the administration of theadditional agent, the anti-Jagged antibody and/or activatableanti-Jagged antibody is administered subsequent to the administration ofthe additional agent, or the anti-Jagged antibody and/or activatableanti-Jagged antibody and the additional agent are administered in analternating fashion. As described herein, the anti-Jagged antibodyand/or activatable anti-Jagged antibody and additional agent areadministered in single doses or in multiple doses.

The invention also provides an isolated nucleic acid molecule encodingan activatable anti-Jagged antibody described herein, as well as vectorsthat include these isolated nucleic acid sequences. The inventionprovides methods of producing an activatable antibody by culturing acell under conditions that lead to expression of the activatableantibody, wherein the cell comprises such a nucleic acid molecule. Insome embodiments, the cell comprises such a vector.

The invention also provides a method of manufacturing activatableantibodies that binds Jagged 1 and Jagged 2 in an activated state by (a)culturing a cell comprising a nucleic acid construct that encodes theactivatable antibody under conditions that lead to expression of theactivatable antibody, wherein the activatable antibody comprises amasking moiety (MM), a cleavable moiety (CM), and an antibody or anantigen binding fragment thereof (AB) that specifically binds Jagged 1and Jagged 2, and (b) recovering the activatable antibody.

The invention also provides a method of manufacturing activatableantibodies that binds Jagged 1 and Jagged 2 in an activated state by (a)culturing a cell comprising a nucleic acid construct that encodes theactivatable antibody under conditions that lead to expression of theactivatable antibody, wherein the activatable antibody comprises amasking moiety (MM), a cleavable moiety (CM), and an antibody or anantigen binding fragment thereof (AB) that specifically binds Jagged 1and Jagged 2, (i) wherein the CM is a polypeptide that includes an aminoacid sequence that functions as a substrate for a protease; and (ii)wherein the CM is positioned in the activatable antibody such that, inan uncleaved state, the MM interferes with specific binding of the AB toJagged 1 and Jagged 2 and in a cleaved state the MM does not interfereor compete with specific binding of the AB to Jagged 1 and Jagged 2; and(b) recovering the activatable antibody.

The present invention also provides methods of treating, preventing,delaying the progression of or otherwise ameliorating a symptom ofpathologies associated with aberrant Jagged 1 and/or Jagged 2 activity(e.g., aberrant signaling, including aberrant signaling through Notchreceptors), or alleviating a symptom associated with such pathologies,by administering an antibody and/or an activatable antibody of theinvention to a subject in which such treatment or prevention is desired.The invention also provides methods of reducing, inhibiting or otherwisemodulating angiogenesis in a subject using the anti-Jagged antibodiesand/or activatable anti-Jagged antibodies described herein. The subjectto be treated is, e.g., human or other mammal. In some embodiments, thesubject is a non-human mammal, such as a non-human primate, companionanimal (e.g., cat, dog, horse), farm animal, work animal, or zoo animal.In some embodiments, the subject is a rodent.

The anti-Jagged antibody and/or activatable anti-Jagged antibody isadministered in an amount sufficient to treat, prevent or alleviate asymptom associated with the pathology. The amount of antibody and/or anactivatable antibody sufficient to treat or prevent the pathology in thesubject is, for example, an amount that is sufficient to reduce Jagged 1and/or Jagged 2 signaling (e.g., Jagged 1 mediated signaling throughNotch receptors and/or Jagged 2 mediated signaling through Notchreceptors). As used herein, the term “reduced” refers to a decreasedsignaling through one or more Notch receptors in the presence of amonoclonal antibody and/or an activatable antibody of the invention.Jagged 1 and/or Jagged 2 mediated signaling is decreased when the levelof signaling through one or more Notch receptors in the presence of ananti-Jagged antibody and/or an activatable antibody of the invention isgreater than or equal to 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,75%, 80%, 90%, 95%, 99%, or 100% lower than a control level of signalingthrough one or more Notch receptors (i.e., the level of signalingthrough one or more Notch receptors in the absence of the monoclonalantibody). Level of signaling through one or more Notch receptors ismeasured using any of a variety of standard techniques, such as, by wayof non-limiting example, measurement of downstream gene activation, suchas Hey and Hes, and/or luciferase reporter assays responsive to Notchreceptor activation. Those skilled in the art will appreciate that thelevel of signaling through one or more Notch receptors can be measuredusing a variety of assays, including, for example, commerciallyavailable kits.

Pathologies treated and/or prevented and/or for which the progression isdelayed and/or for which a symptom is ameliorated using the anti-Jaggedantibodies, activatable anti-Jagged antibodies and/or conjugatedactivatable anti-Jagged antibodies of the invention include, forexample, cancer. In some embodiments, the anti-Jagged antibodies,activatable anti-Jagged antibodies and/or conjugated activatableanti-Jagged antibodies of the invention are used to treat, prevent,delay the progression of, and/or ameliorate a symptom of a pathologysuch as, for example, leukemias, including T-cell acute lymphoblasticleukemia (T-ALL), lymphoblastic diseases including multiple myeloma, andsolid tumors, including lung, colorectal, prostate, pancreatic andbreast, including triple negative breast cancer. In addition, sincenotch signaling is important for the survival and growth of cancer stemcells, inhibition of Jagged dependent notch signaling would impact stemcell growth and survival.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, bone diseaseor metastasis in cancer, regardless of primary tumor origin.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, breast cancer,including by way of non-limiting example, ER/PR+ breast cancer, Her2+breast cancer, triple-negative breast cancer.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, colorectalcancer.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, gastriccancer.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, glioblastoma.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, head and neckcancer.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, lung cancer,such as by way of non-limiting example, non-small cell lung cancer.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, multiplemyeloma.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, ovariancancer.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, pancreaticcancer.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, prostatecancer.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, sarcoma.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, renal cancer,such as by way of nonlimiting example, renal cell carcinoma.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, skin cancer,such as by way of nonlimiting example, squamous cell cancer, basal cellcarcinoma, melanoma.

In addition to cancer, Jagged-dependent notch signaling is critical toepithelial and fibroblast differentiation to myofibroblasts, cells witha central role in the development of fibrotic disease. Inhibition ofJagged dependent notch signaling, and therefore inhibition of theemergence of myofibroblasts, would be an effective treatment forfibrotic diseases of the kidney, liver, lung, and skin. In someembodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies are usedto treat a fibrotic disorder, such as idiopathic pulmonary fibrosis(IPF).

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, fibroticdisease.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, idiopathicpulmonary fibrosis, kidney fibrotic disease, liver fibrotic disease,peritoneal dialysis-induced fibrosis, scleroderma.

In some embodiments, the anti-Jagged antibodies, activatable anti-Jaggedantibodies and/or conjugated activatable anti-Jagged antibodies of theinvention are used to treat, prevent, delay the progression of, and/orameliorate a symptom of a pathology such as, for example, hearing loss.

An anti-Jagged antibody, activatable anti-Jagged antibody and/orconjugated activatable anti-Jagged antibody used in any of theembodiments of these methods and uses can be administered at any stageof the disease. For example, such an anti-Jagged antibody, activatableanti-Jagged antibody and/or conjugated activatable anti-Jagged antibodycan be administered to a patient suffering cancer of any stage, fromearly to metastatic. The terms subject and patient are usedinterchangeably herein.

An anti-Jagged antibody, activatable anti-Jagged antibody and/orconjugated activatable anti-Jagged antibody used in any of theembodiments of these methods and uses can be used in a treatment regimencomprising neoadjuvant therapy.

An anti-Jagged antibody, activatable anti-Jagged antibody and/orconjugated activatable anti-Jagged antibody used in any of theembodiments of these methods and uses can be administered either aloneor in combination with one or more chemotherapeutic agents or otherbiologics.

The invention also provides methods and kits for using the anti-Jaggedantibodies and/or activatable anti-Jagged antibodies in a variety ofdiagnostic and/or prophylactic indications. For example, the inventionprovides methods and kits for detecting presence or absence of acleaving agent and a target of interest in a subject or a sample by (i)contacting a subject or sample with an activatable anti-Jagged antibody,wherein the activatable anti-Jagged antibody comprises a masking moiety(MM), a cleavable moiety (CM) that is cleaved by the cleaving agent, andan antigen binding domain or fragment thereof (AB) that specificallybinds the target of interest, wherein the activatable anti-Jaggedantibody in an uncleaved, non-activated state comprises a structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of theAB to the Jagged target, and wherein the MM does not have an amino acidsequence of a naturally occurring binding partner of the AB and is not amodified form of a natural binding partner of the AB; and (b) wherein,in an uncleaved, non-activated state, the MM interferes with specificbinding of the AB to the Jagged target, and in a cleaved, activatedstate the MM does not interfere or compete with specific binding of theAB to the Jagged target; and (ii) measuring a level of activatedactivatable anti-Jagged antibody in the subject or sample, wherein adetectable level of activated activatable anti-Jagged antibody in thesubject or sample indicates that the cleaving agent and a Jagged targetare present in the subject or sample and wherein no detectable level ofactivated activatable anti-Jagged antibody in the subject or sampleindicates that the cleaving agent, a Jagged target or both the cleavingagent and a Jagged target are absent and/or not sufficiently present inthe subject or sample.

In some embodiments, the activatable anti-Jagged antibody is anactivatable anti-Jagged antibody to which a therapeutic agent isconjugated. In some embodiments, the activatable anti-Jagged antibody isnot conjugated to an agent. In some embodiments, the activatableanti-Jagged antibody comprises a detectable label. In some embodiments,the detectable label is positioned on the AB. In some embodiments,measuring the level of activatable anti-Jagged antibody in the subjector sample is accomplished using a secondary reagent that specificallybinds to the activated antibody, wherein the reagent comprises adetectable label. In some embodiments, the secondary reagent is anantibody comprising a detectable label.

In some embodiments of these methods and kits, the activatableanti-Jagged antibody includes a detectable label. In some embodiments ofthese methods and kits, the detectable label includes an imaging agent,a contrasting agent, an enzyme, a fluorescent label, a chromophore, adye, one or more metal ions, or a ligand-based label. In someembodiments of these methods and kits, the imaging agent comprises aradioisotope. In some embodiments of these methods and kits, theradioisotope is indium or technetium. In some embodiments of thesemethods and kits, the contrasting agent comprises iodine, gadolinium oriron oxide. In some embodiments of these methods and kits, the enzymecomprises horseradish peroxidase, alkaline phosphatase, orβ-galactosidase. In some embodiments of these methods and kits, thefluorescent label comprises yellow fluorescent protein (YFP), cyanfluorescent protein (CFP), green fluorescent protein (GFP), modified redfluorescent protein (mRFP), red fluorescent protein tdimer2 (RFPtdimer2), HCRED, or a europium derivative. In some embodiments of thesemethods and kits, the luminescent label comprises an N-methylacrydiumderivative. In some embodiments of these methods, the label comprises anAlexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In someembodiments of these methods and kits, the ligand-based label comprisesbiotin, avidin, streptavidin or one or more haptens.

In some embodiments of these methods and kits, the subject is a mammal.In some embodiments of these methods and kits, the subject is a human.In some embodiments, the subject is a non-human mammal, such as anon-human primate, companion animal (e.g., cat, dog, horse), farmanimal, work animal, or zoo animal. In some embodiments, the subject isa rodent.

In some embodiments of these methods, the method is an in vivo method.In some embodiments of these methods, the method is an in situ method.In some embodiments of these methods, the method is an ex vivo method.In some embodiments of these methods, the method is an in vitro method.

In some embodiments of the methods and kits, the method or kit is usedto identify or otherwise refine a patient population suitable fortreatment with an activatable anti-Jagged antibody of the disclosure.For example, patients that test positive for both the target (e.g.,Jagged 1 and/or Jagged 2) and a protease that cleaves the substrate inthe cleavable moiety (CM) of the activatable anti-Jagged antibody beingtested in these methods are identified as suitable candidates fortreatment with such an activatable anti-Jagged antibody comprising sucha CM. Likewise, patients that test negative for both of the targets(e.g., Jagged 1 and Jagged 2) and the protease that cleaves thesubstrate in the CM in the activatable antibody being tested using thesemethods might be identified as suitable candidates for another form oftherapy.

In some embodiments, a method or kit is used to identify or otherwiserefine a patient population suitable for treatment with an anti-Jaggedactivatable antibody and/or conjugated activatable anti-Jagged antibody(e.g., activatable antibody to which a therapeutic agent is conjugated)of the disclosure, followed by treatment by administering thatactivatable anti-Jagged antibody and/or conjugated activatableanti-Jagged antibody to a subject in need thereof. For example, patientsthat test positive for both the targets (e.g., Jagged 1 and Jagged 2)and a protease that cleaves the substrate in the cleavable moiety (CM)of the activatable anti-Jagged antibody and/or conjugated activatableanti-Jagged antibody being tested in these methods are identified assuitable candidates for treatment with such antibody and/or such aconjugated activatable anti-Jagged antibody comprising such a CM, andthe patient is then administered a therapeutically effective amount ofthe activatable anti-Jagged antibody and/or conjugated activatableanti-Jagged antibody that was tested. Likewise, patients that testnegative for either or both of the target (e.g., Jagged 1 and/or Jagged2) and the protease that cleaves the substrate in the CM in theactivatable anti-Jagged antibody being tested using these methods mightbe identified as suitable candidates for another form of therapy.

In some embodiments, such patients can be tested with other antibodyand/or conjugated activatable anti-Jagged antibody until a suitableantibody and/or conjugated activatable anti-Jagged antibody fortreatment is identified (e.g., an activatable anti-Jagged antibodyand/or conjugated activatable anti-Jagged antibody comprising a CM thatis cleaved by the patient at the site of disease). In some embodiments,the patient is then administered a therapeutically effective amount ofthe activatable anti-Jagged antibody and/or conjugated for which thepatient tested positive.

In some embodiments of these methods and kits, the MM is a peptidehaving a length from about 4 to 40 amino acids. In some embodiments ofthese methods and kits, the activatable anti-Jagged antibody comprises alinker peptide, wherein the linker peptide is positioned between the MMand the CM. In some embodiments of these methods and kits, theactivatable anti-Jagged antibody comprises a linker peptide, where thelinker peptide is positioned between the AB and the CM. In someembodiments of these methods and kits, the activatable anti-Jaggedantibody comprises a first linker peptide (L1) and a second linkerpeptide (L2), wherein the first linker peptide is positioned between theMM and the CM and the second linker peptide is positioned between the ABand the CM. In some embodiments of these methods and kits, each of L1and L2 is a peptide of about 1 to 20 amino acids in length, and whereineach of L1 and L2 need not be the same linker In some embodiments ofthese methods and kits, one or both of L1 and L2 comprises aglycine-serine polymer. In some embodiments of these methods and kits,at least one of L1 and L2 comprises an amino acid sequence selected fromthe group consisting of (GS)n, (GSGGS)n (SEQ ID NO: 123) and (GGGS)n(SEQ ID NO: 124), where n is an integer of at least one. In someembodiments of these methods and kits, at least one of L1 and L2comprises an amino acid sequence having the formula (GGS)n, where n isan integer of at least one. In some embodiments of these methods andkits, at least one of L1 and L2 comprises an amino acid sequenceselected from the group consisting of Gly-Gly-Ser-Gly (SEQ ID NO: 125),Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 126), Gly-Ser-Gly-Ser-Gly (SEQ ID NO:127), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 128), Gly-Gly-Gly-Ser-Gly (SEQ IDNO: 129), and Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 130).

In some embodiments of these methods and kits, the AB comprises anantibody or antibody fragment sequence selected from the cross-reactiveanti-Jagged antibody sequences presented herein. In some embodiments ofthese methods and kits, the AB comprises a Fab fragment, a scFv or asingle chain antibody (scAb).

In some embodiments of these methods and kits, the cleaving agent is aprotease that is co-localized in the subject or sample with the Jaggedtarget and the CM is a polypeptide that functions as a substrate for theprotease, wherein the protease cleaves the CM in the activatableanti-Jagged antibody when the activatable anti-Jagged antibody isexposed to the protease. In some embodiments of these methods and kits,the CM is a polypeptide of up to 15 amino acids in length. In someembodiments of these methods and kits, the CM is coupled to theN-terminus of the AB. In some embodiments of these methods and kits, theCM is coupled to the C-terminus of the AB. In some embodiments of thesemethods and kits, the CM is coupled to the N-terminus of a VL chain ofthe AB.

In some embodiments of these methods and kits, the cleaving agent is anenzyme and the CM is a substrate for the enzyme. In some embodiments ofthese methods and kits, the enzyme is a protease disclosed herein. Insome embodiments of these methods and kits, the protease is one of theproteases disclosed herein. In some embodiments of these methods andkits, the protease is selected from the group consisting of uPA,legumain, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12,MMP-13, and MMP-14. In some embodiments, the protease is a cathepsin.

Pharmaceutical compositions according to the invention can include anantibody and/or an activatable antibody of the invention and a carrier.These pharmaceutical compositions can be included in kits, such as, forexample, diagnostic kits.

One skilled in the art will appreciate that the antibodies of theinvention have a variety of uses. For example, the proteins of theinvention are used as therapeutic agents to prevent the activation ofJagged-mediated signaling through Notch receptors in a variety ofdisorders. The antibodies of the invention are also used as reagents indiagnostic kits or as diagnostic tools, or these antibodies can be usedin competition assays to generate therapeutic reagents.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph depicting the binding of Anti-Jagged antibodiesreferred to herein as Anti-Jagged 13 and Anti-Jagged 32 to human andmouse Jagged 1 and human Jagged 2.

FIG. 2 is a graph depicting the ability of Anti-Jagged 13 andAnti-Jagged 32 to inhibit Jagged1 binding to human Notch 1.

FIG. 3 is a graph depicting the ability of an anti-Jagged antibodyreferred to herein as 4D11 (also referred to herein as anti-Jagged 4D11,anti-Jagged 4D11 antibody, 4D11 antibody, or antibody 4D11) to inhibitthe growth of BxPC3 xenograft tumors.

FIG. 4 is a graph depicting weight loss by mice administered anti-Jagged4D11.

FIG. 5 is a graph depicting serum concentration of TSLP in miceadministered anti-Jagged 4D11.

FIG. 6 is a graph depicting the ability of the anti-Jagged 4D11 antibodyto inhibit the growth of BxPC3 xenograft tumors for more than 30 dayspost-inoculation.

FIG. 7 is a graph depicting weight loss by mice administered anti-Jagged4D11.

FIG. 8 is a graph depicting the ability of the anti-Jagged 4D11 antibodyto inhibit the proliferation of the multiple myeloma cell line RPMI 8226in co-cultures with human bone marrow.

FIGS. 9A, 9B and 9C are a series of photographs depicting the effect ofanti-Jagged antibodies on rat fibroblast cell line NRK-49F in thepresence or absence of TGFβ1. FIG. 9A demonstrates that cultures ofNRK-F49 retain a characteristic monolayer when cultured in the presenceof 100 nM anti-Jagged 4D11. FIG. 9B demonstrates characteristic fociformation for NRK-F49 cultured in the presence of 10 ng/mL TGFβ1. FIG.9C demonstrates that TGFβ1-stimulated, fibrotic foci formation iscompletely inhibited by 100 nM anti-Jagged 4D11 in cultures treated with10 ng/mL TGFβ1.

FIG. 10 is a series of illustrations depicting the results of screeninga random peptide library in the presence of the anti-Jagged 4D11antibody and Fab fragment. The screening consisted of one round of MACSand two rounds of FACS sorting. The positive population from the secondFACS round was verified to be inhibited by recombinant Jagged proteinfrom binding to the anti-Jagged 4D11 antibody and Fab.

FIG. 11 is a graph that compares binding of anti-Jagged masking moietyJS4896 to binding of affinity matured anti-Jagged masking moietiesJS5340, JS5342, JS5347, and JS5358.

FIG. 12 is a series of graphs depicting the ability of MM 5342 toinhibit binding of activatable anti-Jagged antibodies and an anti-Jaggedmasked antibody to a Jagged target in an in vitro binding ELISA.

FIG. 13 is a photograph and a table depicting proteolytic activation ofactivatable anti-Jagged antibodies.

FIG. 14 is a graph depicting that activatable anti-Jagged antibodiesinhibited the growth of BxPC-3 xenograft tumors in mice, as didanti-Jagged antibody 4D11 (parental antibody). The graph is plotted astumor volume versus number of days post initial dose.

FIG. 15 is a graph depicting a comparison of weight loss of mice thatwere administered activatable anti-Jagged antibodies, masked antibody,or parental antibody.

FIG. 16A is a graph depicting the serum levels of mouse TSLP, where theserum level of mouse TSLP was quantified for individual mice before eachdose and 10 days after the final dose from each group and was thenaveraged. FIG. 16B depicts a time course of TSLP serum concentrationsfor anti-Jagged antibody 4D11 and activatable anti-Jagged antibody5342-1204-4D11.

FIG. 17 compares the average human IgG levels over time in the sera ofmice administered activatable anti-Jagged antibody 5342-1204-4D11 oranti-Jagged antibody 4D11.

FIG. 18 is a graph depicting on cell expression normalized binding tothe anti-Jagged Fab 4D11.

FIG. 19 is a schematic overview of in situ imaging of an activatableantibody: 1. A tissue section is laid over the slide. 2. The slide iscovered with solution containing labeled activatable antibody andincubated. 3. After extensive washing, binding of activated antibody isvisualized.

FIG. 20 is a series of images depicting the abilities of activatableanti-Jagged antibodies 5342-1204-4D11 and 5342-PLGL-4D11 to be activatedand to bind BxPC3 xenograft tumor tissue as demonstrated using in situimaging. The activatable antibodies were labeled with Alexa Fluor® 680to produce labeled activatable antibodies 5342-1204-4D11-AF680 and5342-PLGL-4D11-AF680, also referred to herein as 1204-4D11-AF680 andPLGL-4D11-AF680, respectively. Also tested was labeled anti-Jaggedparental antibody 4D11-AF680. Each of 4D11-AF680 (column 1, row 1),1204-4D11-AF680 (col. 2, row 1) and PLGL-4D11-AF680 (col. 3, row 1) wasincubated with a frozen BxPC3 xenograft tumor tissue sample. The panelsin row 2 represent the fluorescent images obtained after incubation of4D11-AF680 (col. 1), 1204-4D11-AF680 (col. 2) and PLGL-4D11-AF680 (col.3) with frozen BxPC3 xenograft tumor tissue pre-treated with a broadspectrum protease inhibitor cocktail.

FIG. 21 is a series of images depicting activation of activatableanti-Jagged antibodies 5342-1204-4D11 and 5342-PLGL-4D11 as demonstratedby in situ imaging of human pancreatic cancer tissue. Each of 4D11-AF680(4D11) (column 1, row 1), 1204-4D11-AF680 (1204) (col. 1, row 2) andPLGL-4D11-AF680 (PLGL) (col. 1, row 3) was incubated with a frozentissue sample isolated from a human patient with pancreatic cancer. Thepanels in Columns 2, 3, and 4, respectively, represent the fluorescentimages obtained after incubation of 4D11-AF680, 1204-4D11-AF680 andPLGL-4D11-AF680 with frozen pancreatic cancer patient tissue pre-treatedantibody A11, an antibody that specifically binds to the active site ofthe MT-SP1 protease, also known as matriptase; (col. 2); with an MMPinhibitor (FIG. 21, col. 3); or with a broad spectrum protease inhibitorcocktail (col. 4).

FIG. 22 is an image and a graph depicting the in vivo imaging of ananti-Jagged antibody. FIG. 22A is an image that provides arepresentation of the labeled 4D11 antibody fluorescence signal 48 hourspost-injection in the BxPC3 tumor xenograft mouse model. FIG. 22B is agraph showing the mean T/N ratio of average radiant efficacy for theantibody 4D11 dose group ±SD.

FIG. 23 is a graph depicting the effect of the anti-Jagged 4D11 antibodyto inhibit the growth of tumors in the BxPC3 xenograft mouse model whenadministered alone or in combination with an additional anti-canceragent, gemcitabine.

FIG. 24 is a graph depicting BxPC3 growth inhibition curves showing theactivities of: the activated (+uPA) and non-activated (untreated or+PBS) activatable anti-Jagged antibody-agent conjugate; activated andnon-activated activatable anti-Jagged antibody; and anti-Jagged andRituxan antibodies and antibody-agent conjugates.

FIG. 25 is a graph depicting that both anti-Jagged antibody 4D11-MMAEand activatable anti-Jagged antibody 5342-1204-4D11-MMAE inhibitedBxPC-3 xenograft tumor growth more effectively than their unconjugatedcounterparts.

FIG. 26 is a graph depicting the weight loss observed in animals dosedwith anti-Jagged antibody 4D11, anti-Jagged antibody-MMAE, activatableanti-Jagged antibody 5342-1204-4D11 or activatable anti-Jaggedantibody-agent conjugate 5342-1204-4D11-MMAE.

FIGS. 27A-27C are a series of tables and graphs depicting thatanti-Jagged antibodies of the disclosure bind Jagged 1 and Jagged 2 withhigh affinity and with human/rodent cross-reactivity. The table in FIG.27A demonstrates the diversity of specificities among anti-Jaggedclones. The table in FIG. 27B demonstrates that the anti-Jagged antibody4D11 has a high affinity for all four Jagged ligands: human Jagged 1(hJAG1), human Jagged 2 (hJAG2), rat Jagged 1 (rJAG1) and rat Jagged 2(rJAG2). The graphs in FIG. 27C demonstrate that both maturedanti-Jagged Fab fragments of the disclosure (top panel) and matured IgGmolecules of the disclosure (bottom panel) inhibit Notch signaling.

FIG. 28 is a graph depicting efficient internalization of an anti-Jaggedantibody of the disclosure by the BxPC3 pancreatic cell line, plotted aspercent internalization over time, particularly as compared tointernalization by the H292 cell line, a human lung carcinoma cell line.Internalization was demonstrated using a method similar to thatdescribed in Gostring L et al, 2010, Int J Oncol 36, 757-763.

FIG. 29 is a graph depicting the ability of the anti-Jagged activatableantibody 5342-1204-4D11 in combination with Gemcitabine to inhibit thegrowth of BxPC-3 xenograft tumors.

FIG. 30 is a graph depicting that the animals dosed with higher doses ofantibody and Gemcitabine showed significant weight loss, but animalsdosed with an activatable anti-Jagged antibody and Gemcitabine showed noweight loss over that of Gemcitabine alone.

FIG. 31 is a graph depicting that only the administration of the 20mg/kg anti-Jagged antibody in combination with Gemcitabine showedelevated serum mTSLP.

FIG. 32 is a graph depicting that the anti-Jagged antibody 4D11 waseffective in limiting the growth of prostate tumors in TRAMP mice.

FIG. 33 is a graph depicting that the anti-Jagged antibody 4D11 potentlyinhibited the growth of spontaneous tumors in Her2/neu transgenic mice.

FIG. 34 is a series of images depicting the feasibility of conducting insitu imaging using non-labeled (i.e., unlabeled) activatable antibodiesand a secondary reagent that comprises a detectable label and thatspecifically binds the AB of the activatable antibody.

FIG. 35 is a series of images depicting the activation of non-labeledanti-Jagged activatable antibody 5342-1204-4D11 by tumor tissues of atransgenic prostate cancer model (TRAMP).

FIG. 36 is a graph depicting the abilities of antibody 4D11, activatableantibody 5342-1204-4D11), and MT-SP1-activated antibody 5342-1204-4D11to bind to human Jagged 1.

FIG. 37 is a graph depicting that the anti-Jagged activatable antibody5342-1204-4D11 inhibited the growth of the H292 xenograft tumors.

FIG. 38 is a graph depicting that the activatable anti-Jagged antibody5342-1204-4D11 showed no elevation in TSLP, while animals that wereadministered the antibody at 6.7 and 20 mg/kg showed increased TSLP ascompared to the IVIg treated group.

FIG. 39 is a series of images depicting that hyperkeratosis was observedin the antibody-treated group, while the activatable antibody-treatedgroup showed limited or no hyperkeratosis.

DETAILED DESCRIPTION

The present invention describes novel compositions for the diagnosis andtreatment of cancer. Specifically this invention provides antibodiesthat bind Jagged 1 and Jagged 2 and inhibit their binding to andsignaling through Notch receptors. The present invention providesmonoclonal antibodies that specifically bind Jagged 1 and Jagged 2(i.e., cross-reactive monoclonal antibodies). These antibodies arecollectively referred to herein as “anti-Jagged antibodies.”

Indications that would benefit from Jagged inhibition would includecancers. For example, indications would include leukemias, includingT-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic diseasesincluding multiple myeloma, and solid tumors, including lung,colorectal, prostate, pancreatic and breast, including triple negativebreast cancer. In addition, since notch signaling is important for thesurvival and growth of cancer stem cells, inhibition of Jagged dependentnotch signaling would impact stem cell growth and survival. For example,indications include bone disease or metastasis in cancer, regardless ofprimary tumor origin; breast cancer, including by way of non-limitingexample, ER/PR+ breast cancer, Her2+ breast cancer, triple-negativebreast cancer; colorectal cancer; gastric cancer; glioblastoma; head andneck cancer; lung cancer, such as by way of non-limiting example,non-small cell lung cancer; multiple myeloma ovarian cancer; pancreaticcancer; prostate cancer; sarcoma; renal cancer, such as by way ofnonlimiting example, renal cell carcinoma; and/or skin cancer, such asby way of nonlimiting example, squamous cell cancer, basal cellcarcinoma, melanoma.

In addition to cancer, Jagged-dependent notch signaling is critical toepithelial and fibroblast differentiation to myofibroblasts, cells witha central role in the development of fibrotic disease. Inhibition ofJagged dependent notch signaling, and therefore inhibition of theemergence of myofibroblasts, would be an effective treatment forfibrotic diseases of the kidney, liver, lung, and skin. For example,indications would include a fibrotic disorder, such as idiopathicpulmonary fibrosis (IPF); kidney fibrotic disease, liver fibroticdisease, peritoneal dialysis-induced fibrosis, and/or scleroderma.

Other suitable indications include, for example, a pathology such as,for example, hearing loss.

The antibodies of the present invention bind to a Jagged 1 epitopeand/or a Jagged 2 epitope with an equilibrium binding constant (K_(d))of ≦1 μM. In some embodiments, antibodies of the present invention bindto a Jagged 1 epitope and/or a Jagged 2 epitope with a K_(d) of ≦100 nM,≦10 nM, or ≦1 nM. In some embodiments, the anti-Jagged antibodiesprovided herein exhibit a K_(d) in the range approximately between ≦1 nMto about 1 pM. In some embodiments, antibodies of the present inventionbind to a Jagged 1 epitope and/or a Jagged 2 epitope with an “off rateconstant” (K_(off)) of <10⁻², <10⁻³, <10⁻⁴, or <10⁻⁵. In someembodiments, anti-Jagged antibodies provided herein exhibit a K_(off) of<10⁻⁴. In some embodiments, anti-Jagged antibodies provided hereinexhibit a K_(off) of <10⁻⁵. In some embodiments, an antibody of thepresent invention binds to an EGF domain of Jagged 1 and of Jagged 2. Insome embodiments, an antibody of the present invention binds to a DSLdomain of Jagged 1 and of Jagged 2.

The anti-Jagged antibodies and/or an activatable antibody of theinvention serve to modulate, block, inhibit, reduce, antagonize,neutralize or otherwise interfere with the biological activity of Jagged1 and/or Jagged 2. Biological activities of Jagged 1 and/or Jagged 2include, for example, signaling through one or more Notch receptors. Forexample, the anti-Jagged antibodies completely or partially inhibitJagged 1 and/or Jagged 2 biological activity by partially or completelymodulating, blocking, inhibiting, reducing antagonizing, neutralizing,or otherwise interfering with the binding of Jagged 1 and/or Jagged toone or more Notch receptors, or otherwise partially or completelymodulating, blocking, inhibiting, reducing, antagonizing, neutralizingJagged 1 and/or Jagged 2 mediated signaling activity.

The anti-Jagged antibodies are considered to completely modulate, block,inhibit, reduce, antagonize, neutralize or otherwise interfere withJagged 1 and/or Jagged 2 biological activity when the level Jagged 1and/or Jagged 2 activity in the presence of the anti-Jagged antibody isdecreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% ascompared to the level of activity in the absence of an anti-Jaggedantibody described herein. The anti-Jagged antibodies are considered topartially modulate, block, inhibit, reduce, antagonize, neutralize orotherwise interfere with Jagged 1 and/or Jagged 2 activity when thelevel Jagged 1 and/or Jagged 2 activity in the presence of theanti-Jagged antibody is decreased by less than 95%, e.g., 10%, 20%, 25%,30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level ofactivity in the absence of an anti-Jagged antibody described herein.Examples of Jagged activity include, but are not limited to, celldivision and differentiation, cell survival/apoptosis,epithelial-mesenchymal transition (EMT) and invasion, angiogenesis,self-renewal of cancer stem cells, and osteolytic bone lesions. Whilenot being bound by theory, it is thought that Jagged's role inangiogenesis is different from that of VEGF.

DEFINITIONS

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” or “immunospecifically bind” is meant that theantibody reacts with one or more antigenic determinants of the desiredantigen and does not react with other polypeptides or binds at muchlower affinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, domain antibody, single chain, Fab,and F(ab′)₂ fragments, scFvs, and an Fab expression library.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, IgG₃, IgG₄, and others.Furthermore, in humans, the light chain may be a kappa chain or a lambdachain.

The term “monoclonal antibody” (mAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. For example, antibodies may be raisedagainst N-terminal or C-terminal peptides of a polypeptide. An antibodyis said to specifically bind an antigen when the dissociation constantis ≦1 μM; for example, in some embodiments ≦100 nM and in someembodiments ≦10 nM.

As used herein, the terms “specific binding,” “immunological binding,”and “immunological binding properties” refer to the non-covalentinteractions of the type that occur between an immunoglobulin moleculeand an antigen for which the immunoglobulin is specific. The strength,or affinity of immunological binding interactions can be expressed interms of the dissociation constant (K_(d)) of the interaction, wherein asmaller K_(d) represents a greater affinity. Immunological bindingproperties of selected polypeptides can be quantified using methods wellknown in the art. One such method entails measuring the rates ofantigen-binding site/antigen complex formation and dissociation, whereinthose rates depend on the concentrations of the complex partners, theaffinity of the interaction, and geometric parameters that equallyinfluence the rate in both directions. Thus, both the “on rate constant”(K_(on)) and the “off rate constant” (K_(off)) can be determined bycalculation of the concentrations and the actual rates of associationand dissociation. (See Nature 361:186-87 (1993)). The ratio ofK_(off)/K_(on) enables the cancellation of all parameters not related toaffinity, and is equal to the dissociation constant K_(d). (See,generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). Anantibody of the present invention is said to specifically bind to EGFR,when the equilibrium binding constant (K_(d)) is ≦1 μM, for example insome embodiments ≦100 nM, in some embodiments ≦10 nM, and in someembodiments ≦100 pM to about 1 pM, as measured by assays such asradioligand binding assays or similar assays known to those skilled inthe art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide that it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the invention include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules shown herein, andnucleic acid molecules encoding the light chain immunoglobulin moleculesshown herein.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of murine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the invention comprise the heavychain immunoglobulin molecules shown herein, and the light chainimmunoglobulin molecules shown herein, as well as antibody moleculesformed by combinations comprising the heavy chain immunoglobulinmolecules with light chain immunoglobulin molecules, such as kappa lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andthat has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences that are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein meansnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term oligonucleotide referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. In some embodiments, oligonucleotides are10 to 60 bases in length, for example in some embodiments 12, 13, 14,15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides areusually single stranded, e.g., for probes, although oligonucleotides maybe double stranded, e.g., for use in the construction of a gene mutant.Oligonucleotides of the invention are either sense or antisenseoligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotide linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such asα-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, andother unconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and thatare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and that are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, for example in some embodiments, at least 90 percentsequence identity, in some embodiments at least 95 percent sequenceidentity, and in some embodiments at least 99 percent sequence identity.

In some embodiments, residue positions that are not identical differ byconservative amino acid substitutions.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, for example in someembodiments at least 80%, 90%, 95%, and in some embodiments 99%. Inparticular, conservative amino acid replacements are contemplated.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids are generally divided into families: (1) acidic amino acidsare aspartate, glutamate; (2) basic amino acids are lysine, arginine,histidine; (3) non-polar amino acids are alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan, and (4)uncharged polar amino acids are glycine, asparagine, glutamine,cysteine, serine, threonine, tyrosine. The hydrophilic amino acidsinclude arginine, asparagine, aspartate, glutamine, glutamate,histidine, lysine, serine, and threonine. The hydrophobic amino acidsinclude alanine, cysteine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan, tyrosine and valine. Other familiesof amino acids include (i) serine and threonine, which are thealiphatic-hydroxy family; (ii) asparagine and glutamine, which are theamide containing family; (iii) alanine, valine, leucine and isoleucine,which are the aliphatic family; and (iv) phenylalanine, tryptophan, andtyrosine, which are the aromatic family. For example, it is reasonableto expect that an isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid will not have a major effect on the binding or properties of theresulting molecule, especially if the replacement does not involve anamino acid within a framework site. Whether an amino acid change resultsin a functional peptide can readily be determined by assaying thespecific activity of the polypeptide derivative. Assays are described indetail herein. Fragments or analogs of antibodies or immunoglobulinmolecules can be readily prepared by those of ordinary skill in the art.In some embodiments, amino- and carboxy-termini of fragments or analogsoccur near boundaries of functional domains. Structural and functionaldomains can be identified by comparison of the nucleotide and/or aminoacid sequence data to public or proprietary sequence databases.Computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal. Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the invention.

In some embodiments, amino acid substitutions are those that: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions (forexample, conservative amino acid substitutions) may be made in thenaturally-occurring sequence (for example, in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion and/or oneor more internal deletion(s), but where the remaining amino acidsequence is identical to the corresponding positions in thenaturally-occurring sequence deduced, for example, from a full lengthcDNA sequence. Fragments typically are at least 5, 6, 8 or 10 aminoacids long, for example in some embodiments at least 14 amino acidslong, in some embodiments at least 20 amino acids long, usually at least50 amino acids long, and in some embodiments at least 70 amino acidslong. The term “analog” as used herein refers to polypeptides that arecomprised of a segment of at least 25 amino acids that has substantialidentity to a portion of a deduced amino acid sequence and that hasspecific binding to EGFR, under suitable binding conditions. Typically,polypeptide analogs comprise a conservative amino acid substitution (oraddition or deletion) with respect to the naturally-occurring sequence.Analogs typically are at least 20 amino acids long, for example in someembodiments at least 50 amino acids long or longer, and can often be aslong as a full-length naturally-occurring polypeptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., a fluorophore, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, for example in some embodiments more than about 85%, 90%,95%, and 99%. In some embodiments, the object species is purified toessential homogeneity (contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

Indications that would benefit from Jagged inhibition include leukemias,including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblasticdiseases including multiple myeloma, and solid tumors, including lung,colorectal, prostate, pancreatic and breast, including triple negativebreast cancer. In addition, since notch signaling is important for thesurvival and growth of cancer stem cells, inhibition of Jagged dependentnotch signaling would impact stem cell growth and survival. In additionto cancer, Jagged dependent notch signaling is critical to epithelialand fibroblast differentiation to myofibroblasts, cells with a centralrole in the development of fibrotic disease. Inhibition of Jaggeddependent notch signaling, and therefore inhibition of the emergence ofmyofibroblasts, would be an effective treatment for fibrotic diseases ofthe kidney, liver, lung, and skin.

Anti-Jagged Antibodies and Activatable Anti-Jagged Antibodies

Monoclonal antibodies and/or an activatable antibody of the inventionhave the ability to inhibit Jagged 1 and/or Jagged 2 mediated signalingthrough Notch receptors. Inhibition is determined using any of a varietyof art-recognized techniques, including the assays described in theexamples provided herein.

Anti-Jagged antibodies and/or activatable anti-Jagged antibodies of theinvention additionally include, for example, the combinations of heavychain complementarity determining regions (VH CDRs) and light chaincomplementarity determining regions (VL CDRs). Examples of such CDRs areshown in Table 2 below or are the CDRs of antibodies disclosed herein,including, but not limited to, those in Table 3 and Table 4. In someembodiments, anti-Jagged antibodies of the invention include antibodiesthat contain a combination of a VH CDR1 sequence, a VH CDR2 sequence, aVH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence of at least one antibody selected from the group consisting ofthe 4D11 antibody, the 4B2 antibody, the 4E7 antibody, the 4E11antibody, the 6B7 antibody, and the 6F8 antibody. In some embodiments,anti-Jagged antibodies of the invention include antibodies that containa combination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequencethat are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the sequences of at least one antibody selected fromthe group consisting of the 4D11 antibody, the 4B2 antibody, the 4E7antibody, the 4E11 antibody, the 6B7 antibody, and the 6F8 antibody.

The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VHCDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein at least one CDR sequence is selected from the groupconsisting of a VH CDR1 sequence that includes at least the amino acidsequence SYAMS (SEQ ID NO: 200); a VH CD2 sequence that includes atleast the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VHCDR3 sequence that includes at least the amino acid sequence DIGGRSAFDY(SEQ ID NO: 209); a VL CDR1 sequence that includes at least the aminoacid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes at least the amino acid sequence AASSLQS (SEQ ID NO: 211); anda VL CDR3 sequence that includes at least the amino acid sequenceQQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VHCDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein at least one CDR sequence is selected from the groupconsisting of a VH CDR1 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence SYAMS (SEQ ID NO: 200); a VH CD2 sequencethat includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequenceSIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VH CDR3 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO:209); a VL CDR1 sequence that includes a sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQID NO: 211); and a VL CDR3 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence QQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a VH CDR1 sequence that includes at least the amino acidsequence SYAMS (SEQ ID NO: 200); a VH CD2 sequence that includes atleast the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VHCDR3 sequence that includes at least the amino acid sequence DIGGRSAFDY(SEQ ID NO: 209); a VL CDR1 sequence that includes at least the aminoacid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes at least the amino acid sequence AASSLQS (SEQ ID NO: 211); anda VL CDR3 sequence that includes at least the amino acid sequenceQQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a VH CDR1 sequence that includes a sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe amino acid sequence SYAMS (SEQ ID NO: 200); a VH CD2 sequence thatincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequenceSIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VH CDR3 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO:209); a VL CDR1 sequence that includes a sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQID NO: 211); and a VL CDR3 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence QQTVVAPPL (SEQ ID NO: 212).

Exemplary antibodies and/or activatable antibodies of the inventioninclude, for example, the 4D11 antibody, the 4B2 antibody, the 4E7antibody, the 4E11 antibody, the 6B7 antibody, and the 6F8 antibody, andvariants thereof. These antibodies show specificity for human Jagged 1and Jagged 2, and they have been shown to inhibit human Jagged 1 and/orhuman Jagged 2 mediated signaling through Notch receptors. Theseantibodies include the combinations of a heavy chain variable region(VH) and a light chain variable region (VL), as shown in the amino acidand corresponding nucleic acid sequences listed in Table 4 and shown inExample 5.

Also included in the invention are antibodies and/or activatableantibodies that bind to the same epitope as the antibodies describedherein. For example, antibodies and/or an activatable antibody of theinvention specifically bind to human Jagged 1, wherein the antibodybinds to an epitope that includes one or more amino acid residues onhuman Jagged 1 (shown, for example, in Accession Nos. AAC52020.1;AAB84053.1; NP_(—)000205.1; P78504.3; AAB39007.1; EAX10341.1;AAI26208.1; AAI26206.1; AAH98393.1; CAC07198.1 and/or BAG35596.1).Antibodies and/or activatable antibodies of the invention specificallybind to human Jagged 2, wherein the antibody binds to an epitope thatincludes one or more amino acid residues on human Jagged 2 (see e.g.,Accession Nos. AAB61285, AAB71189.1, EAW81901.1, NP_(—)002217.3, and/orQ9Y219.3). Antibodies of the invention specifically bind both humanJagged 1 and human Jagged 2, wherein the antibody binds to an epitopethat includes one or more amino acid residues on human Jagged 1 and anepitope that includes one or more amino acid residues on human Jagged 2.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a monoclonal antibody(e.g., fully human monoclonal antibody) and/or an activatable antibodyhas the same or similar specificity as a monoclonal antibody and/or anactivatable antibody of the invention (e.g., 4D11, 4B2, 4E7, 4E11, 6B7,and/or 6F8 antibodies and activatable antibodies that include theseantibodies) by ascertaining whether the former prevents the latter frombinding to Jagged 1, Jagged 2 or both Jagged 1 and Jagged 2. If themonoclonal antibody and/or an activatable antibody being tested competeswith the monoclonal antibody and/or an activatable antibody of theinvention, as shown by a decrease in binding by the monoclonal antibodyand/or an activatable antibody of the invention, then the two monoclonalantibodies and/or activatable antibodies bind to the same, or a closelyrelated, epitope.

One embodiment for determining whether a monoclonal antibody and/or anactivatable antibody has the specificity of monoclonal antibody and/oran activatable antibody of the invention is to pre-incubate themonoclonal antibody and/or an activatable antibody of the invention withsoluble Jagged 1 and/or Jagged 2 protein (with which it is normallyreactive), and then add the monoclonal antibody and/or an activatableantibody being tested to determine if the monoclonal antibody and/or anactivatable antibody being tested is inhibited in its ability to bindJagged 1 and/or Jagged 2. If the monoclonal antibody and/or anactivatable antibody being tested is inhibited then, in all likelihood,it has the same, or functionally equivalent, epitopic specificity as themonoclonal antibody and/or an activatable antibody of the invention.

Screening of monoclonal antibodies and/or an activatable antibody of theinvention, can be also carried out, e.g., by measuring Jagged 1 and/orJagged 2 mediated signaling through Notch receptors and determiningwhether the test monoclonal antibody is able to modulate, block,inhibit, reduce, antagonize, neutralize or otherwise interfere withJagged 1 and/or Jagged 2 mediated signaling through Notch receptors.Examples of Jagged activity include, but are not limited to, celldivision and differentiation, cell survival/apoptosis,epithelial-mesenchymal transition (EMT) and invasion, angiogenesis,self-renewal of cancer stem cells, and osteolytic bone lesions. Methodsto measure such activities are known to those skilled in the art.

Various procedures known within the art may be used for the productionof monoclonal antibodies directed against human Jagged 1 and/or humanJagged 2, or against derivatives, fragments, analogs homologs ororthologs thereof. (See, for example, Antibodies: A Laboratory Manual,Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., incorporated herein by reference). Fully humanantibodies are antibody molecules in which the entire sequence of boththe light chain and the heavy chain, including the CDRs, arise fromhuman genes. Such antibodies are termed “human antibodies,” or “fullyhuman antibodies” herein. Human monoclonal antibodies are prepared, forexample, using the procedures described in the Examples provided below.Human monoclonal antibodies can be also prepared by using the triomatechnique; the human B-cell hybridoma technique (see Kozbor, et al.,1983 Immunol Today 4: 72); and the EBV hybridoma technique to producehuman monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Humanmonoclonal antibodies may be utilized and may be produced by using humanhybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96).

Antibodies are purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen that is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

The antibodies of the invention are monoclonal antibodies. Monoclonalantibodies that modulate, block, inhibit, reduce, antagonize, neutralizeor otherwise interfere with Jagged 1 and/or Jagged 2 mediated signalingthrough Notch receptors are generated, e.g., by immunizing an animalwith Jagged 1 and/or Jagged 2, such as, for example, murine, rat orhuman Jagged 1 and/or Jagged 2 or an immunogenic fragment, derivative orvariant thereof. Alternatively, the animal is immunized with cellstransfected with a vector containing a nucleic acid molecule encodingJagged 1 and/or Jagged 2 such that Jagged 1 and/or Jagged 2 is expressedand associated with the surface of the transfected cells. Alternatively,the antibodies are obtained by screening a library that containsantibody or antigen binding domain sequences for binding to Jagged 1and/or Jagged 2. This library is prepared, e.g., in bacteriophage asprotein or peptide fusions to a bacteriophage coat protein that isexpressed on the surface of assembled phage particles and the encodingDNA sequences contained within the phage particles (i.e., “phagedisplayed library”). Hybridomas resulting from myeloma/B cell fusionsare then screened for reactivity to Jagged 1 and Jagged 2.

Monoclonal antibodies are prepared, for example, using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that, in some embodiments,contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

In some embodiments, immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. In some embodiments, immortalized cell lines are murinemyeloma lines, which can be obtained, for instance, from the SalkInstitute Cell Distribution Center, San Diego, Calif. and the AmericanType Culture Collection, Manassas, Va. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofmonoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. The binding specificity of monoclonal antibodies produced bythe hybridoma cells is determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem., 107:220 (1980). Moreover, in therapeutic applications ofmonoclonal antibodies, it is important to identify antibodies having ahigh degree of specificity and a high binding affinity for the targetantigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Once isolated, the DNA canbe placed into expression vectors, which are then transfected into hostcells such as simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. For antibodies isolated from murine hybridomas, the DNA also canbe modified, for example, by substituting the coding sequence for humanheavy and light chain constant domains in place of the homologous murinesequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13(1994)). Antibody-encoding DNA can be covalently joined to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

Human Antibodies and Humanization of Antibodies

Monoclonal antibodies of the invention include fully human antibodies orhumanized antibodies. These antibodies are suitable for administrationto humans without engendering an immune response by the human againstthe administered immunoglobulin.

An anti-Jagged antibody is generated, for example, using the proceduresdescribed in the Examples provided below.

In some methods, an anti-Jagged antibody is developed, for example,using phage-display methods using antibodies containing only humansequences. Such approaches are well-known in the art, e.g., inWO92/01047 and U.S. Pat. No. 6,521,404, which are hereby incorporated byreference. In this approach, a combinatorial library of phage carryingrandom pairs of light and heavy chains are screened using natural orrecombinant source of Jagged 1, Jagged 2 and/or both Jagged 1 and Jagged2 or fragments thereof. In another approach, an anti-Jagged antibody canbe produced by a process wherein at least one step of the processincludes immunizing a transgenic, non-human animal with human Jagged 1protein, Jagged 2 protein or both Jagged 1 and Jagged 2 protein. In thisapproach, some of the endogenous heavy and/or kappa light chain loci ofthis xenogenic non-human animal have been disabled and are incapable ofthe rearrangement required to generate genes encoding immunoglobulins inresponse to an antigen. In addition, at least one human heavy chainlocus and at least one human light chain locus have been stablytransfected into the animal. Thus, in response to an administeredantigen, the human loci rearrange to provide genes encoding humanvariable regions immunospecific for the antigen. Upon immunization,therefore, the xenomouse produces B-cells that secrete fully humanimmunoglobulins.

A variety of techniques are well-known in the art for producingxenogenic non-human animals. For example, see U.S. Pat. No. 6,075,181and No. 6,150,584, which is hereby incorporated by reference in itsentirety. This general strategy was demonstrated in connection withgeneration of the first XenoMouse™ strains as published in 1994. SeeGreen et al. Nature Genetics 7:13-21 (1994), which is herebyincorporated by reference in its entirety. See also, U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2 and EuropeanPatent No., EP 0 463 151 B1 and International Patent Applications No. WO94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and related familymembers.

In an alternative approach, others have utilized a “minilocus” approachin which an exogenous Ig locus is mimicked through the inclusion ofpieces (individual genes) from the Ig locus. Thus, one or more VH genes,one or more D_(H) genes, one or more J_(H) genes, a mu constant region,and a second constant region (e.g., a gamma constant region) are formedinto a construct for insertion into an animal. See e.g., U.S. Pat. Nos.5,545,806; 5,545,807; 5,591,669; 5,612,205;5,625,825; 5,625,126;5,633,425; 5,643,763; 5,661,016; 5,721,367; 5,770,429; 5,789,215;5,789,650; 5,814,318; 5,877; 397; 5,874,299; 6,023,010; and 6,255,458;and European Patent No. 0 546 073 B1; and International PatentApplication Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO98/24884 and related family members.

Generation of human antibodies from mice in which, through microcellfusion, large pieces of chromosomes, or entire chromosomes, have beenintroduced, has also been demonstrated. See European Patent ApplicationNos. 773 288 and 843 961.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. When chimericantibodies have a human constant region and a murine variable region, itis expected that certain human anti-chimeric antibody (HACA) responseswill be observed, particularly in chronic or multi-dose utilizations ofthe antibody. Thus, it would be desirable to provide fully humanantibodies against Jagged 1, Jagged 2 and/or both Jagged 1 and Jagged 2in order to vitiate or otherwise mitigate concerns and/or effects ofHAMA or HACA response.

The production of antibodies with reduced immunogenicity is alsoaccomplished via humanization, chimerization and display techniquesusing appropriate libraries. It will be appreciated that murineantibodies or antibodies from other species can be humanized orprimatized using techniques well known in the art. See e.g., Winter andHarris Immunol Today 14:43 46 (1993) and Wright et al. Crit, Reviews inImmunol. 12125-168 (1992). The antibody of interest may be engineered byrecombinant DNA techniques to substitute the CH1, CH2, CH3, hingedomains, and/or the framework domain with the corresponding humansequence (See WO 92102190 and U.S. Pat. Nos. 5,530,101, 5,585,089,5,693,761, 5,693,792, 5,714,350, and 5,777,085). Also, the use of IgcDNA for construction of chimeric immunoglobulin genes is known in theart (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521(1987)). mRNA is isolated from a hybridoma or other cell producing theantibody and used to produce cDNA. The cDNA of interest may be amplifiedby the polymerase chain reaction using specific primers (U.S. Pat. Nos.4,683,195 and 4,683,202). Alternatively, a library is made and screenedto isolate the sequence of interest. The DNA sequence encoding thevariable region of the antibody is then fused to human constant regionsequences. The sequences of human constant regions genes may be found inKabat et al. (1991) Sequences of Proteins of immunological Interest,N.I.H. publication no. 91-3242. Human C region genes are readilyavailable from known clones. The choice of isotype will be guided by thedesired effecter functions, such as complement fixation, or activity inantibody-dependent cellular cytotoxicity. Suitable isotypes are IgG₁,IgG₃ and IgG₄. Either of the human light chain constant regions, kappaor lambda, may be used. The chimeric antibody is then expressed byconventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g., by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting antibody may be joined to anystrong promoter, including retroviral LTRs, e.g., SV-40 early promoter,(Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcoma virus LTR(Gorman et al. P.N.A.S. 79:6777 (1982)), and moloney murine leukemiavirus LTR (Grosschedl et al. Cell 41:885 (1985)). Also, as will beappreciated, native Ig promoters and the like may be used.

Further, human antibodies or antibodies from other species can begenerated through display type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes andPlückthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley andSmith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol.17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russel etal. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol.Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A(1992), and U.S. Pat. No. 5,733,743. If display technologies areutilized to produce antibodies that are not human, such antibodies canbe humanized as described above.

Using these techniques, antibodies can be generated to Jagged 1, Jagged2 and/or both Jagged 1 and Jagged 2 expressing cells, Jagged 1 itself,Jagged 2 itself, forms of Jagged 1 and/or Jagged 2, epitopes or peptidesthereof, and expression libraries thereto (See e.g., U.S. Pat. No.5,703,057) that can thereafter be screened as described above for theactivities described herein.

Anti-Jagged antibodies of the invention can be expressed by a vectorcontaining a DNA segment encoding the single chain antibody describedabove.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA,gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, that have a targeting moiety (e.g. a ligand toa cellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vectors (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US 95/02140 (WO 95/22618) that is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Suitable vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.In some embodiments, DNA viral vectors are preferred. These vectorsinclude pox vectors such as orthopox or avipox vectors, herpesvirusvectors such as a herpes simplex I virus (HSV) vector (see Geller, A. I.et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning:Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England)(1995); Geller, A. I. et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603(1993); Geller, A. I., et al., Proc Natl. Acad. Sci USA 87:1149 (1990),Adenovirus Vectors (see LeGal LaSalle et al., Science, 259:988 (1993);Davidson, et al., Nat. Genet 3:219 (1993); Yang, et al., J. Virol.69:2004 (1995) and Adeno-associated Virus Vectors (see Kaplitt, M. G. etal., Nat. Genet. 8:148 (1994).

Pox viral vectors introduce the gene into the cell's cytoplasm. Avipoxvirus vectors result in only short term expression of the nucleic acid.In some embodiments, adenovirus vectors, adeno-associated virus vectorsand herpes simplex virus (HSV) vectors are preferred for introducing thenucleic acid into neural cells. The adenovirus vector results in shorterterm expression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icy) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

These vectors can be used to express large quantities of antibodies thatcan be used in a variety of ways, for example, to detect the presence ofJagged 1, Jagged 2 and/or both Jagged 1 and Jagged 2 in a sample. Theantibody can also be used to try to bind to and disrupt Jagged 1, Jagged2, and/or both Jagged 1 and Jagged 2-related signaling.

Techniques can be adapted for the production of single-chain antibodiesspecific to an antigenic protein of the invention (see e.g., U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof F_(ab) expression libraries (see e.g., Huse, et al., 1989 Science246: 1275-1281) to allow rapid and effective identification ofmonoclonal F_(ab) fragments with the desired specificity for a proteinor derivatives, fragments, analogs or homologs thereof. Antibodyfragments that contain the idiotypes to a protein antigen may beproduced by techniques known in the art including, but not limited to:(i) an F_((ab′)2) fragment produced by pepsin digestion of an antibodymolecule; (ii) an F_(ab) fragment generated by reducing the disulfidebridges of an F_((ab′)2) fragment; (iii) an F_(ab) fragment generated bythe treatment of the antibody molecule with papain and a reducing agentand (iv) F_(v) fragments.

The invention also includes F_(v), F_(ab), F_(ab′) and F_((ab′)2)antibody fragments, single chain anti-Jagged antibodies, bispecificanti-Jagged antibodies, multispecific anti-Jagged antibodies, andheteroconjugate anti-Jagged antibodies.

Bispecific antibodies are antibodies that have binding specificities forat least two different antigens. In the present case, one of the bindingspecificities is for Jagged 1 and Jagged 2. The second binding target isany other antigen, and advantageously is a cell-surface protein orreceptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of different antibody molecules,of which only one has the correct bispecific structure. The purificationof the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion is, in some embodiments, with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers that are recovered from recombinant cellculture. The interface comprises at least a part of the CH3 region of anantibody constant domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker that is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (scFv, or ScFv) dimers has alsobeen reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm that binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular antigen. Bispecific antibodies can alsobe used to direct cytotoxic agents to cells that express a particularantigen. These antibodies possess an antigen-binding arm and an arm thatbinds a cytotoxic agent or a radionuclide chelator, such as EOTUBE,DPTA, DOTA, or TETA. Another bispecific antibody embodiment binds theprotein antigen described herein and further binds tissue factor (TF).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (see U.S. Pat. No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating diseases and disorders associated with Jagged 1and/or Jagged 2 signaling. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively,an antibody can be engineered that has dual Fc regions and can therebyhave enhanced complement lysis and ADCC capabilities. (See Stevenson etal., Anti-Cancer Drug Design, 3: 219-230 (1989)).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate). Suitablecytotoxic agents include, for example, dolastatins and derivativesthereof (e.g. auristatin E, AFP, MMAF, MMAE). For example, the cytotoxicagent is monomethyl auristatin E (MMAE). In some embodiments, the agentis an agent selected from the group listed in Table 30. In someembodiments, the agent is a dolastatin. In some embodiments, the agentis an auristatin or derivative thereof. In some embodiments, the agentis auristatin E or a derivative thereof. In some embodiments, the agentis monomethyl auristatin E (MMAE). In some embodiments, the agent is amaytansinoid or maytansinoid derivative. In some embodiments, the agentis DM1 or DM4. In some embodiments, the agent is a duocarmycin orderivative thereof. In some embodiments, the agent is a calicheamicin orderivative thereof.

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ⁶⁴Cu, ¹²⁵I, ¹³¹I, ¹³¹In, ^(99m)Tc, ⁹⁰Y, ¹⁸⁶Re, and ⁸⁹Zr.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Table 30 lists some of the exemplary pharmaceutical agents that may beemployed in the herein described invention but in no way is meant to bean exhaustive list.

TABLE 30 Exemplary Pharmaceutical Agents for Conjugation CYTOTOXICAGENTS Auristatins Auristatin E Monomethyl auristatin E (MMAE) Desmethylauristatin E (DMAE) Auristatin F Monomethyl auristatin F (MMAF)Desmethyl auristatin F (DMAF) Auristatin derivatives, e.g., amidesthereof Auristatin tyramine Auristatin quinoline Dolastatins Dolastatinderivatives Dolastatin 16 DmJ Dolastatin 16 Dpv Maytansinoids, e.g.DM-1; DM-4 Maytansinoid derivatives Duocarmycin Duocarmycin derivativesAlpha-amanitin Anthracyclines Doxorubicin Daunorubicin BryostatinsCamptothecin Camptothecin derivatives 7-substituted Camptothecin 10,11-Difluoromethylenedioxycamptothecin Combretastatins DebromoaplysiatoxinKahalalide-F Discodermolide Ecteinascidins ANTIVIRALS Acyclovir Vira ASymmetrel ANTIFUNGALS Nystatin Turbostatin Phenstatins HydroxyphenstatinSpongistatin 5 Spongistatin 7 Halistatin 1 Halistatin 2 Halistatin 3Modified Bryostatins Halocomstatins Pyrrolobenzimidazoles (PBI)Cibrostatin6 Doxaliform Anthracyclins analogues Anthracyclins analoguesCemadotin analogue (CemCH2-SH) Pseudomonas toxin A (PE38) variantPseudomonas toxin A (ZZ-PE38) variant ZJ-101 OSW-14-Nitrobenzyloxycarbonyl Derivatives of O6-Benzylguanine Topoisomeraseinhibitors Hemiasterlin Cephalotaxine HomoharringtoninePyrrolobenzodiazepine dimers (PBDs) Functionalizedpyrrolobenzodiazepenes Calicheamicins Podophyllotoxins Taxanes Vincaalkaloids CONJUGATABLE DETECTION REAGENTS Fluorescein and derivativesthereof Fluorescein isothiocyanate (FITC) ADDITIONAL ANTI-NEOPLASTICSAdriamycin Cerubidine Bleomycin Alkeran Velban Oncovin FluorouracilMethotrexate Thiotepa Bisantrene Novantrone Thioguanine ProcarabizineCytarabine ANTI-BACTERIALS Aminoglycosides Streptomycin NeomycinKanamycin Amikacin Gentamicin Tobramycin Streptomycin B SpectinomycinAmpicillin Sulfanilamide Polymyxin Chloramphenicol RADIOPHARMACEUTICALS¹²⁵I ¹³¹I ⁸⁹Zr ¹¹¹In ¹²³I ¹³¹I ⁹⁹mTc ²⁰¹Tl ¹³³Xe ¹¹C ⁶²Cu ¹⁸F ⁶⁸Ga ¹³N¹⁵O ³⁸K ⁸²Rb ⁹⁹mTc (Technetium) HEAVY METALS Barium Gold PlatinumANTI-MYCOPLASMALS Tylosine Spectinomycin

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies of theinvention. (See, for example, “Conjugate Vaccines”, Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),Carger Press, New York, (1989), the entire contents of which areincorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. In some embodiments,the preferred binding is, however, covalent binding. Covalent bindingcan be achieved either by direct condensation of existing side chains orby the incorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987).

Suitable linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat.No. 5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker Particularlysuitable linkers include: (i) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (ii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); and (iii) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.#2165-G.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, the linker SMPT contains a sterically hindereddisulfide bond, and can form conjugates with increased stability.Disulfide linkages, are in general, less stable than other linkagesbecause the disulfide linkage is cleaved in vitro, resulting in lessconjugate available.

The reagent EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride is useful to create a carboxamide starting with acarboxylic acid and a primary or secondary amine. Thus, EDC may be usedto link lysine residues in an antibody with a carboxylic acid in alinker or toxin, or to link aspartate or glutamate residues in anantibody with an amine in a linker or toxin. Such conjugation reactionsutilizing EDC may be enhanced by addition of NHS (N-hydroxysuccinimide)or sulfo-NHS (N-hydroxy-3-oxysulfonylsuccinimide). Addition of NHS orsulfo-NHS to such conjugation reactions may enhance the rate,completeness, selectivity, and/or reproducibility of the conjugationreactions.

In some embodiments, the linkers are cleavable. In some embodiments, thelinkers are non-cleavable. In some embodiments, two or more linkers arepresent. The two or more linkers are all the same, e.g., cleavable ornon-cleavable, or the two or more linkers are different, e.g., at leastone cleavable and at least one non-cleavable.

The present invention utilizes several methods for attaching agents toABs: (a) attachment to the carbohydrate moieties of the AB, or (b)attachment to sulfhydryl groups of the AB, or (c) attachment to aminogroups of the AB, or (d) attachment to carboxylate groups of the AB.According to the invention, ABs may be covalently attached to an agentthrough an intermediate linker having at least two reactive groups, oneto react with AB and one to react with the agent. The linker, which mayinclude any compatible organic compound, can be chosen such that thereaction with AB (or agent) does not adversely affect AB reactivity andselectivity. Furthermore, the attachment of linker to agent might notdestroy the activity of the agent. Suitable linkers for reaction withoxidized antibodies or oxidized antibody fragments include thosecontaining an amine selected from the group consisting of primary amine,secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine,semicarbazide and thiosemicarbazide groups. Such reactive functionalgroups may exist as part of the structure of the linker, or may beintroduced by suitable chemical modification of linkers not containingsuch groups.

According to the present invention, suitable linkers for attachment toreduced ABs include those having certain reactive groups capable ofreaction with a sulfhydryl group of a reduced antibody or fragment. Suchreactive groups include, but are not limited to: reactive haloalkylgroups (including, for example, haloacetyl groups), p-mercuribenzoategroups and groups capable of Michael-type addition reactions (including,for example, maleimides and groups of the type described by Mitra andLawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

According to the present invention, suitable linkers for attachment toneither oxidized nor reduced Abs include those having certain functionalgroups capable of reaction with the primary amino groups present inunmodified lysine residues in the Ab. Such reactive groups include, butare not limited to, NHS carboxylic or carbonic esters, sulfo-NHScarboxylic or carbonic esters, 4-nitrophenyl carboxylic or carbonicesters, pentafluorophenyl carboxylic or carbonic esters, acylimidazoles, isocyanates, and isothiocyanates.

According to the present invention, suitable linkers for attachment toneither oxidized nor reduced Abs include those having certain functionalgroups capable of reaction with the carboxylic acid groups present inaspartate or glutamate residues in the Ab, which have been activatedwith suitable reagents. Suitable activating reagents include EDC, withor without added NHS or sulfo-NHS, and other dehydrating agents utilizedfor carboxamide formation. In these instances, the functional groupspresent in the suitable linkers would include primary and secondaryamines, hydrazines, hydroxylamines, and hydrazides.

The agent may be attached to the linker before or after the linker isattached to the AB. In certain applications it may be desirable to firstproduce an AB-linker intermediate in which the linker is free of anassociated agent. Depending upon the particular application, a specificagent may then be covalently attached to the linker In other embodimentsthe AB is first attached to the MM, CM and associated linkers and thenattached to the linker for conjugation purposes.

Branched Linkers:

In specific embodiments, branched linkers that have multiple sites forattachment of agents are utilized. For multiple site linkers, a singlecovalent attachment to an AB would result in an AB-linker intermediatecapable of binding an agent at a number of sites. The sites may bealdehyde or sulfhydryl groups or any chemical site to which agents canbe attached.

Alternatively, higher specific activity (or higher ratio of agents toAB) can be achieved by attachment of a single site linker at a pluralityof sites on the AB. This plurality of sites may be introduced into theAB by either of two methods. First, one may generate multiple aldehydegroups and/or sulfhydryl groups in the same AB. Second, one may attachto an aldehyde or sulfhydryl of the AB a “branched linker” havingmultiple functional sites for subsequent attachment to linkers. Thefunctional sites of the branched linker or multiple site linker may bealdehyde or sulfhydryl groups, or may be any chemical site to whichlinkers may be attached. Still higher specific activities may beobtained by combining these two approaches, that is, attaching multiplesite linkers at several sites on the AB.

Cleavable Linkers:

Peptide linkers that are susceptible to cleavage by enzymes of thecomplement system, such as but not limited to urokinase, tissueplasminogen activator, trypsin, plasmin, or another enzyme havingproteolytic activity may be used in one embodiment of the presentinvention. According to one method of the present invention, an agent isattached via a linker susceptible to cleavage by complement. Theantibody is selected from a class that can activate complement. Theantibody-agent conjugate, thus, activates the complement cascade andreleases the agent at the target site. According to another method ofthe present invention, an agent is attached via a linker susceptible tocleavage by enzymes having a proteolytic activity such as a urokinase, atissue plasminogen activator, plasmin, or trypsin. These cleavablelinkers are useful in conjugated activatable antibodies that include anextracellular toxin, e.g., by way of non-limiting example, any of theextracellular toxins shown in Table 30.

Non-liming examples of cleavable linker sequences are provided in Table31.

TABLE 31 Exemplary Linker Sequences for Conjugation Types of CleavableSequences Amino Acid Sequence Plasmin cleavable sequences Pro-urokinasePRFKIIGG (SEQ ID NO: 235) PRFRIIGG (SEQ ID NO: 236) TGFβ SSRHRRALD (SEQID NO: 237) Plasminogen RKSSIIIRMRDVVL (SEQ ID NO: 238) StaphylokinaseSSSFDKGKYKKGDDA (SEQ ID NO: 239) SSSFDKGKYKRGDDA (SEQ ID NO: 240) FactorXa cleavable sequences IEGR (SEQ ID NO: 241) IDGR (SEQ ID NO: 242)GGSIDGR (SEQ ID NO: 243) MMP cleavable sequences Gelatinase A PLGLWA(SEQ ID NO: 244) Collagenase cleavable sequences Calf skin collagen(α1(I) chain) GPQGIAGQ (SEQ ID NO: 245) Calf skin collagen (α2(I) chain)GPQGLLGA (SEQ ID NO: 246) Bovine cartilage collagen (α1(II) chain) GIAGQ(SEQ ID NO: 247) Human liver collagen (α1(III) chain) GPLGIAGI (SEQ IDNO: 248) Human α₂M GPEGLRVG (SEQ ID NO: 249) Human PZP YGAGLGVV (SEQ IDNO: 250) AGLGVVER (SEQ ID NO: 251) AGLGISST (SEQ ID NO: 252) Rat α₁MEPQALAMS (SEQ ID NO: 253) QALAMSAI (SEQ ID NO: 254) Rat α₂M AAYHLVSQ(SEQ ID NO: 255) MDAFLESS (SEQ ID NO: 256) Rat α₁I₃(2J) ESLPVVAV (SEQ IDNO: 257) Rat α₁I₃(27J) SAPAVESE (SEQ ID NO: 258) Human fibroblastcollagenase DVAQFVLT (SEQ ID NO: 259) (autolytic cleavages) VAQFVLTE(SEQ ID NO: 260) AQFVLTEG (SEQ ID NO: 261) PVQPIGPQ (SEQ ID NO: 262)

In addition, agents may be attached via disulfide bonds (for example,the disulfide bonds on a cysteine molecule) to the AB. Since many tumorsnaturally release high levels of glutathione (a reducing agent) this canreduce the disulfide bonds with subsequent release of the agent at thesite of delivery. In certain specific embodiments the reducing agentthat would modify a CM would also modify the linker of the conjugatedactivatable antibody.

Spacers and Cleavable Elements:

In still another embodiment, it may be necessary to construct the linkerin such a way as to optimize the spacing between the agent and the AB ofthe activatable antibody. This may be accomplished by use of a linker ofthe general structure:

W—(CH₂)n-Q

whereinW is either —NH—CH₂— or —CH₂—;Q is an amino acid, peptide; andn is an integer from 0 to 20.

In still other embodiments, the linker may comprise a spacer element anda cleavable element. The spacer element serves to position the cleavableelement away from the core of the AB such that the cleavable element ismore accessible to the enzyme responsible for cleavage. Certain of thebranched linkers described above may serve as spacer elements.

Throughout this discussion, it should be understood that the attachmentof linker to agent (or of spacer element to cleavable element, orcleavable element to agent) need not be particular mode of attachment orreaction. Any reaction providing a product of suitable stability andbiological compatibility is acceptable.

Serum Complement and Selection of Linkers:

According to one method of the present invention, when release of anagent is desired, an AB that is an antibody of a class that can activatecomplement is used. The resulting conjugate retains both the ability tobind antigen and activate the complement cascade. Thus, according tothis embodiment of the present invention, an agent is joined to one endof the cleavable linker or cleavable element and the other end of thelinker group is attached to a specific site on the AB. For example, ifthe agent has an hydroxy group or an amino group, it may be attached tothe carboxy terminus of a peptide, amino acid or other suitably chosenlinker via an ester or amide bond, respectively. For example, suchagents may be attached to the linker peptide via a carbodimide reaction.If the agent contains functional groups that would interfere withattachment to the linker, these interfering functional groups can beblocked before attachment and deblocked once the product conjugate orintermediate is made. The opposite or amino terminus of the linker isthen used either directly or after further modification for binding toan AB that is capable of activating complement.

Linkers (or spacer elements of linkers) may be of any desired length,one end of which can be covalently attached to specific sites on the ABof the activatable antibody. The other end of the linker or spacerelement may be attached to an amino acid or peptide linker

Thus when these conjugates bind to antigen in the presence of complementthe amide or ester bond that attaches the agent to the linker will becleaved, resulting in release of the agent in its active form. Theseconjugates, when administered to a subject, will accomplish delivery andrelease of the agent at the target site, and are particularly effectivefor the in vivo delivery of pharmaceutical agents, antibiotics,antimetabolites, antiproliferative agents and the like as presented inbut not limited to those in Table 30.

Linkers for Release without Complement Activation:

In yet another application of targeted delivery, release of the agentwithout complement activation is desired since activation of thecomplement cascade will ultimately lyse the target cell. Hence, thisapproach is useful when delivery and release of the agent should beaccomplished without killing the target cell. Such is the goal whendelivery of cell mediators such as hormones, enzymes, corticosteroids,neurotransmitters, genes or enzymes to target cells is desired. Theseconjugates may be prepared by attaching the agent to an AB that is notcapable of activating complement via a linker that is mildly susceptibleto cleavage by serum proteases. When this conjugate is administered toan individual, antigen-antibody complexes will form quickly whereascleavage of the agent will occur slowly, thus resulting in release ofthe compound at the target site.

Biochemical Cross Linkers:

In other embodiments, the activatable antibody may be conjugated to oneor more therapeutic agents using certain biochemical cross-linkers.Cross-linking reagents form molecular bridges that tie togetherfunctional groups of two different molecules. To link two differentproteins in a step-wise manner, hetero-bifunctional cross-linkers can beused that eliminate unwanted homopolymer formation.

Peptidyl linkers cleavable by lysosomal proteases are also useful, forexample, Val-Cit, Val-Ala or other dipeptides. In addition, acid-labilelinkers cleavable in the low-pH environment of the lysosome may be used,for example: bis-sialyl ether. Other suitable linkers includecathepsin-labile substrates, particularly those that show optimalfunction at an acidic pH.

Exemplary hetero-bifunctional cross-linkers are referenced in Table 32.

TABLE 32 Exemplary Hetero-Bifunctional Cross Linkers HETERO-BIFUNCTIONALCROSS-LINKERS Spacer Arm Length after Reactive Advantages cross-linkingLinker Toward and Applications (Angstroms) SMPT Primary amines Greaterstability 11.2 Å Sulfhydryls SPDP Primary amines Thiolation 6.8 ÅSulfhydryls Cleavable cross-linking LC-SPDP Primary amines Extendedspacer arm 15.6 Å Sulfhydryls Sulfo-LC- Primary amines Extender spacerarm 15.6 Å SPDP Sulfhydryls Water-soluble SMCC Primary amines Stablemaleimide reactive 11.6 Å group Sulfhydryls Enzyme-antibody conjugationHapten-carrier protein conjugation Sulfo- Primary amines Stablemaleimide reactive 11.6 Å SMCC group Sulfhydryls Water-solubleEnzyme-antibody conjugation MBS Primary amines Enzyme-antibodyconjugation 9.9 Å Sulfhydryls Hapten-carrier protein conjugation Sulfo-Primary amines Water-soluble 9.9 Å MBS Sulfhydryls SIAB Primary aminesEnzyme-antibody conjugation 10.6 Å Sulfhydryls Sulfo- Primary aminesWater-soluble 10.6 Å SIAB Sulfhydryls SMPB Primary amines Extendedspacer arm 14.5 Å Sulfhydryls Enzyme-antibody conjugation Sulfo- Primaryamines Extended spacer arm 14.5 Å SMPB Sulfhydryls Water-soluble EDE/Primary amines Hapten-Carrier conjugation 0 Sulfo- Carboxyl NHS groupsABH Carbohydrates Reacts with sugar groups 11.9 Å Nonselective

Non-Cleavable Linkers or Direct Attachment:

In still other embodiments of the invention, the conjugate may bedesigned so that the agent is delivered to the target but not released.This may be accomplished by attaching an agent to an AB either directlyor via a non-cleavable linker

These non-cleavable linkers may include amino acids, peptides, D-aminoacids or other organic compounds that may be modified to includefunctional groups that can subsequently be utilized in attachment to ABsby the methods described herein. A-general formula for such an organiclinker could be

W—(CH₂)n-Q

whereinW is either —NH—CH₂— or —CH₂—;Q is an amino acid, peptide; andn is an integer from 0 to 20.

Non-Cleavable Conjugates:

Alternatively, a compound may be attached to ABs that do not activatecomplement. When using ABs that are incapable of complement activation,this attachment may be accomplished using linkers that are susceptibleto cleavage by activated complement or using linkers that are notsusceptible to cleavage by activated complement.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Activatable Anti-Jagged Antibodies

The activatable antibodies and activatable antibody compositionsprovided herein contain at least an antibody or antibody fragmentthereof (collectively referred to as AB throughout the disclosure), thatspecifically binds Jagged 1 and Jagged 2, wherein the AB is modified bya masking moiety (MM).

When the AB is modified with a MM and is in the presence of Jagged 1and/or Jagged 2, specific binding of the AB to its target is reduced orinhibited, as compared to the specific binding of the AB not modifiedwith an MM or the specific binding of the parental AB to the target.

The K_(d) of the AB modified with a MM towards the target, i.e., Jagged1 and Jagged 2, is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500,5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000,10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000,10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000,100-1,000,100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000,1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000,10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000,or 100,000-10,000,000 times greater than the K_(d) of the AB notmodified with an MM or the parental AB towards the target. Conversely,the binding affinity of the AB modified with a MM towards the target,i.e., Jagged 1 and Jagged 2, is at least 5, 10, 25, 50, 100, 250, 500,1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100,10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000,10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or100,000-10,000,000 times lower than the binding affinity of the AB notmodified with an MM or the parental AB towards the target.

The dissociation constant (K_(d)) of the MM towards the AB is generallygreater than the K_(d) of the AB towards the target, i.e., Jagged 1 andJagged 2. The K_(d) of the MM towards the AB can be at least 5, 10, 25,50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 oreven 10,000,000 times greater than the K_(d) of the AB towards thetarget, i.e., Jagged 1 and Jagged 2. Conversely, the binding affinity ofthe MM towards the AB is generally lower than the binding affinity ofthe AB towards the target, i.e., Jagged 1 and Jagged 2. The bindingaffinity of MM towards the AB can be at least 5, 10, 25, 50, 100, 250,500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000times lower than the binding affinity of the AB towards the target,i.e., Jagged 1 and Jagged 2.

When the AB is modified with a MM and is in the presence of the target,i.e., Jagged 1 and Jagged 2, specific binding of the AB to its target isreduced or inhibited, as compared to the specific binding of the AB notmodified with an MM or the specific binding of the parental AB to thetarget. When compared to the binding of the AB not modified with an MMor the binding of the parental AB to the target, i.e., Jagged 1 andJagged 2, the AB's ability to bind the target when modified with an MMcan be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24,30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120,150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months ormore when measured in vivo or in an in vitro assay.

The MM inhibits the binding of the AB to the target, i.e., Jagged 1 andJagged 2. The MM binds the antigen binding domain of the AB and inhibitsbinding of the AB to Jagged 1 and Jagged 2. The MM can stericallyinhibit the binding of the AB to the target, i.e., Jagged 1 and Jagged2. The MM can allosterically inhibit the binding of the AB to itstarget. In these embodiments when the AB is modified or coupled to a MMand in the presence of target, i.e., Jagged 1 and Jagged 2, there is nobinding or substantially no binding of the AB to the target, or no morethan 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, or 50% binding of the AB to the target, ascompared to the binding of the AB not modified with an MM, the parentalAB, or the AB not coupled to an MM to the target, for at least 2, 4, 6,8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30,45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 months or longer when measured in vivo or in an in vitro assay.

When an AB is coupled to or modified by a MM, the MM ‘masks’ or reducesor otherwise inhibits the specific binding of the AB to Jagged 1 andJagged 2. When an AB is coupled to or modified by a MM, such coupling ormodification can effect a structural change that reduces or inhibits theability of the AB to specifically bind its target.

An AB coupled to or modified with an MM can be represented by thefollowing formulae (in order from an amino (N) terminal region tocarboxyl (C) terminal region:

(MM)-(AB)

(AB)-(MM)

(MM)-L-(AB)

(AB)-L-(MM)

where MM is a masking moiety, the AB is an antibody or antibody fragmentthereof, and the L is a linker. In many embodiments, it may be desirableto insert one or more linkers, e.g., flexible linkers, into thecomposition so as to provide for flexibility.

In certain embodiments, the MM is not a natural binding partner of theAB. In some embodiments the MM contains no or substantially no homologyto any natural binding partner of the AB. In other embodiments the MM isno more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or 80% similar to any natural binding partner of the AB.In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to anynatural binding partner of the AB. In some embodiments, the MM is nomore than 25% identical to any natural binding partner of the AB. Insome embodiments, the MM is no more than 50% identical to any naturalbinding partner of the AB. In some embodiments, the MM is no more than20% identical to any natural binding partner of the AB. In someembodiments, the MM is no more than 10% identical to any natural bindingpartner of the AB.

In some embodiments, the activatable antibodies include an AB that ismodified by an MM and also includes one or more cleavable moieties (CM).Such activatable antibodies exhibit activatable/switchable binding, tothe AB's target, i.e., Jagged 1 and Jagged 2. Activatable antibodiesgenerally include an antibody or antibody fragment (AB), modified by orcoupled to a masking moiety (MM) and a modifiable or cleavable moiety(CM). In some embodiments, the CM contains an amino acid sequence thatserves as a substrate for a protease of interest.

The elements of the activatable antibodies are arranged so that the MMand CM are positioned such that in a cleaved (or relatively active)state and in the presence of a target, the AB binds a target, i.e.,Jagged 1 and Jagged 2, while in an uncleaved (or relatively inactive)state in the presence of the target, specific binding of the AB to itstarget, i.e., Jagged 1 and Jagged 2, is reduced or inhibited. Thespecific binding of the AB to its target can be reduced due to theinhibition or masking of the AB's ability to specifically bind itstarget by the MM.

The K_(d) of the AB modified with a MM and a CM towards the target,i.e., Jagged 1 and Jagged 2, is at least 5, 10, 25, 50, 100, 250, 500,1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100,10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000,10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or100,000-10,000,000 times greater than the K_(d) of the AB not modifiedwith an MM and a CM or the parental AB towards the target, i.e., Jagged1 and Jagged 2. Conversely, the binding affinity of the AB modified witha MM and a CM towards the target, i.e., Jagged 1 and Jagged 2, is atleast 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000,100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 orgreater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000,10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000,100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000,1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000,10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lowerthan the binding affinity of the AB not modified with an MM and a CM orthe parental AB towards the target, i.e., Jagged 1 and Jagged 2.

When the AB is modified with a MM and a CM and is in the presence of thetarget but not in the presence of a modifying agent (for example aprotease), specific binding of the AB to its target, i.e., Jagged 1 andJagged 2, is reduced or inhibited, as compared to the specific bindingof the AB not modified with an MM and a CM or the parental AB to thetarget. When compared to the binding of the parental AB or the bindingof an AB not modified with an MM and a CM to its target, the AB'sability to bind the target when modified with an MM and a CM can bereduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36,48, 60, 72, 84, or 96 hours or 5, 10, 15, 30, 45, 60, 90, 120, 150, or180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longerwhen measured in vivo or in an in vitro assay.

As used herein, the term cleaved state refers to the condition of theactivatable antibodies following modification of the CM by a protease.The term uncleaved state, as used herein, refers to the condition of theactivatable antibodies in the absence of cleavage of the CM by aprotease. As discussed above, the term “activatable antibodies” is usedherein to refer to an activatable antibody in both its uncleaved(native) state, as well as in its cleaved state. It will be apparent tothe ordinarily skilled artisan that in some embodiments a cleavedactivatable antibody may lack an MM due to cleavage of the CM byprotease, resulting in release of at least the MM (e.g., where the MM isnot joined to the activatable antibodies by a covalent bond (e.g., adisulfide bond between cysteine residues).

By activatable or switchable is meant that the activatable antibodyexhibits a first level of binding to a target, i.e., Jagged 1 and/orJagged 2, when in a inhibited, masked or uncleaved state (i.e., a firstconformation), and a second level of binding to the target, i.e., Jagged1 and/or Jagged 2, in the uninhibited, unmasked and/or cleaved state(i.e., a second conformation), where the second level of target bindingis greater than the first level of binding. In general, the access oftarget to the AB of the activatable antibody is greater in the presenceof a cleaving agent capable of cleaving the CM than in the absence ofsuch a cleaving agent. Thus, when the activatable antibody is in theuncleaved state, the AB is inhibited from target binding and can bemasked from target binding (i.e., the first conformation is such the ABcannot bind the target), and in the cleaved state the AB is notinhibited or is unmasked to target binding.

The CM and AB of the activatable antibodies are selected so that the ABrepresents a binding moiety for Jagged 1 and Jagged 2, and the CMrepresents a substrate for a protease that is co-localized with Jagged 1and Jagged 2 at a treatment site or diagnostic site in a subject. Theactivatable antibodies disclosed herein find particular use where, forexample, a protease capable of cleaving a site in the CM is present atrelatively higher levels in target-containing tissue of a treatment siteor diagnostic site than in tissue of non-treatment sites (for example inhealthy tissue).

In some embodiments activatable antibodies provide for reduced toxicityand/or adverse side effects that could otherwise result from binding ofthe AB at non-treatment sites if the AB were not masked or otherwiseinhibited from binding Jagged 1 and Jagged 2.

In general, an activatable antibody can be designed by selecting an ABof interest and constructing the remainder of the activatable antibodyso that, when conformationally constrained, the MM provides for maskingof the AB or reduction of binding of the AB to its target. Structuraldesign criteria can be to be taken into account to provide for thisfunctional feature.

Activatable antibodies exhibiting a switchable phenotype of a desireddynamic range for target binding in an inhibited versus an uninhibitedconformation are provided. Dynamic range generally refers to a ratio of(a) a maximum detected level of a parameter under a first set ofconditions to (b) a minimum detected value of that parameter under asecond set of conditions. For example, in the context of an activatableantibody, the dynamic range refers to the ratio of (a) a maximumdetected level of target protein, i.e., Jagged 1 and Jagged 2, bindingto an activatable antibody in the presence of protease capable ofcleaving the CM of the activatable antibodies to (b) a minimum detectedlevel of target protein, i.e., Jagged 1 and Jagged 2, binding to anactivatable antibody in the absence of the protease. The dynamic rangeof an activatable antibody can be calculated as the ratio of theequilibrium dissociation constant of an activatable antibody cleavingagent (e.g., enzyme) treatment to the equilibrium dissociation constantof the activatable antibodies cleaving agent treatment. The greater thedynamic range of an activatable antibody, the better the switchablephenotype of the activatable antibody. Activatable antibodies havingrelatively higher dynamic range values (e.g., greater than 1) exhibitmore desirable switching phenotypes such that target protein binding bythe activatable antibodies occurs to a greater extent (e.g.,predominantly occurs) in the presence of a cleaving agent (e.g., enzyme)capable of cleaving the CM of the activatable antibodies than in theabsence of a cleaving agent.

Activatable antibodies can be provided in a variety of structuralconfigurations. Exemplary formulae for activatable antibodies areprovided below. It is specifically contemplated that the N- toC-terminal order of the AB, MM and CM may be reversed within anactivatable antibody. It is also specifically contemplated that the CMand MM may overlap in amino acid sequence, e.g., such that the CM iscontained within the MM.

For example, activatable antibodies can be represented by the followingformula (in order from an amino (N) terminal region to carboxyl (C)terminal region:

(MM)-(CM)-(AB)

(AB)-(CM)-(MM)

where MM is a masking moiety, CM is a cleavable moiety, and AB is anantibody or fragment thereof. It should be noted that although MM and CMare indicated as distinct components in the formulae above, in allexemplary embodiments (including formulae) disclosed herein it iscontemplated that the amino acid sequences of the MM and the CM couldoverlap, e.g., such that the CM is completely or partially containedwithin the MM. In addition, the formulae above provide for additionalamino acid sequences that may be positioned N-terminal or C-terminal tothe activatable antibodies elements.

In certain embodiments, the MM is not a natural binding partner of theAB. In some embodiments the MM contains no or substantially no homologyto any natural binding partner of the AB. In other embodiments the MM isno more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or 80% similar to any natural binding partner of the AB.In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to anynatural binding partner of the AB. In some embodiments, the MM is nomore than 50% identical to any natural binding partner of the AB. Insome embodiments, the MM is no more than 25% identical to any naturalbinding partner of the AB. In some embodiments, the MM is no more than20% identical to any natural binding partner of the AB. In someembodiments, the MM is no more than 10% identical to any natural bindingpartner of the AB.

In many embodiments it may be desirable to insert one or more linkers,e.g., flexible linkers, into the activatable antibody construct so as toprovide for flexibility at one or more of the MM-CM junction, the CM-ABjunction, or both. For example, the AB, MM, and/or CM may not contain asufficient number of residues (e.g., Gly, Ser, Asp, Asn, especially Glyand Ser, particularly Gly) to provide the desired flexibility. As such,the switchable phenotype of such activatable antibody constructs maybenefit from introduction of one or more amino acids to provide for aflexible linker. In addition, as described below, where the activatableantibody is provided as a conformationally constrained construct, aflexible linker can be operably inserted to facilitate formation andmaintenance of a cyclic structure in the uncleaved activatable antibody.

For example, in certain embodiments an activatable antibody comprisesone of the following formulae (where the formula below represent anamino acid sequence in either N- to C-terminal direction or C- toN-terminal direction):

(MM)-L1-(CM)-(AB)

(MM)-(CM)-L2-(AB)

(MM)-L1-(CM)-L2-(AB)

wherein MM, CM, and AB are as defined above; wherein L1 and L2 are eachindependently and optionally present or absent, are the same ordifferent flexible linkers that include at least 1 flexible amino acid(e.g., Gly). In addition, the formulae above provide for additionalamino acid sequences that may be positioned N-terminal or C-terminal tothe activatable antibodies elements. Examples include, but are notlimited to, targeting moieties (e.g., a ligand for a receptor of a cellpresent in a target tissue) and serum half-life extending moieties(e.g., polypeptides that bind serum proteins, such as immunoglobulin(e.g., IgG) or serum albumin (e.g., human serum albumin (HAS)).

In some embodiments, the cleavable moiety (CM) of the activatableantibody includes an amino acid sequence that can serve as a substratefor a protease, usually an extracellular protease. The CM may beselected based on a protease that is co-localized in tissue with thedesired target of the AB of the activatable antibody. A variety ofdifferent conditions are known in which a target of interest isco-localized with a protease, where the substrate of the protease isknown in the art. In the example of cancer, the target tissue can be acancerous tissue, particularly cancerous tissue of a solid tumor. Thereare reports in the literature of increased levels of proteases havingknown substrates in a number of cancers, e.g., solid tumors. See, e.g.,La Rocca et al, (2004) British J. of Cancer 90(7): 1414-1421. Non-limingexamples of disease include: all types of cancers (breast, lung,colorectal, prostate, melanomas, head and neck, pancreatic, etc.),rheumatoid arthritis, Crohn's disuse, SLE, cardiovascular damage,ischemia, etc. For example, indications would include leukemias,including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblasticdiseases including multiple myeloma, and solid tumors, including lung,colorectal, prostate, pancreatic and breast, including triple negativebreast cancer. For example, indications include bone disease ormetastasis in cancer, regardless of primary tumor origin; breast cancer,including by way of non-limiting example, ER/PR+ breast cancer, Her2+breast cancer, triple-negative breast cancer; colorectal cancer; gastriccancer; glioblastoma; head and neck cancer; lung cancer, such as by wayof non-limiting example, non-small cell lung cancer; multiple myelomaovarian cancer; pancreatic cancer; prostate cancer; sarcoma; renalcancer, such as by way of nonlimiting example, renal cell carcinoma;and/or skin cancer, such as by way of nonlimiting example, squamous cellcancer, basal cell carcinoma, melanoma. In addition to cancer,Jagged-dependent notch signaling is critical to epithelial andfibroblast differentiation to myofibroblasts, cells with a central rolein the development of fibrotic disease. Inhibition of Jagged dependentnotch signaling, and therefore inhibition of the emergence ofmyofibroblasts, would be an effective treatment for fibrotic diseases ofthe kidney, liver, lung, and skin. For example, indications wouldinclude a fibrotic disorder, such as idiopathic pulmonary fibrosis(IPF); kidney fibrotic disease, liver fibrotic disease, peritonealdialysis-induced fibrosis, and/or scleroderma. Other suitableindications include, for example, a pathology such as, for example,hearing loss.

The CM is specifically cleaved by an enzyme at a rate of about0.001-1500×10⁴ M⁻¹S⁻¹ or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1,2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750,1000, 1250, or 1500×10⁴ M⁻¹S⁻¹.

For specific cleavage by an enzyme, contact between the enzyme and CM ismade. When the activatable antibody comprising an AB coupled to a MM anda CM is in the presence of target and sufficient enzyme activity, the CMcan be cleaved. Sufficient enzyme activity can refer to the ability ofthe enzyme to make contact with the CM and effect cleavage. It canreadily be envisioned that an enzyme may be in the vicinity of the CMbut unable to cleave because of other cellular factors or proteinmodification of the enzyme.

Exemplary substrates include but are not limited to substrates cleavableby one or more of the following enzymes or proteases in Table 33:

ADAMS, ADAMTS, e.g. ADAM8 ADAM9 ADAM10 ADAM12 ADAM15 ADAM17/TACE ADAMTS1ADAMTS4 ADAMTS5 Aspartate proteases, e.g., BACE Aspartic cathepsins,e.g., Cathepsin D Cathepsin E Caspases, e.g., Caspase 1 Caspase 2Caspase 3 Caspase 4 Caspase 5 Caspase 6 Caspase 7 Caspase 8 Caspase 9Caspase 10 Caspase 14 Cysteine cathepsins, e.g., Cathepsin B Cathepsin CCathepsin K Cathepsin L Cathepsin S Cathepsin V/L2 Cathepsin X/Z/PCysteine proteinases, e.g., Cruzipain Legumain KLKs, e.g., KLK4 KLK5KLK6 KLK7 KLK8 KLK10 KLK11 KLK13 KLK14 Metallo proteinases, e.g., MeprinNeprilysin PSMA BMP-1 MMPs, e.g., MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9MMP-10 MMP-11 MMP-12 MMP-13 MMP-14 MMP-15 MMP-19 MMP-23 MMP-24 MMP-26MMP-27 Serine proteases, e.g., activated protein C Cathepsin A CathepsinG Chymase coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa,FXIIa) Elastase Granzyme B Guanidinobenzoatase Human Neutrophil ElastaseNS3/4A Plasmin PSA tPA Thrombin Tryptase uPA Type II TransmembraneSerine Proteases (TTSPs), e.g., DESC1 DPP-4 FAP Hepsin Matriptase-2MT-SP1/Matriptase TMPRSS2 TMPRSS3 TMPRSS4

For example, in some embodiments, the substrate is cleavable by one ormore of the following enzymes or proteases: uPA, legumain, MT-SP1,ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12, MMP-13, and/or MMP-14.In some embodiments, the protease is selected from the group of uPA,legumain, and MT-SP1. In some embodiments, the protease is a matrixmetalloproteinase.

Linkers suitable for use in compositions described herein are generallyones that provide flexibility of the modified AB or the activatableantibodies to facilitate the inhibition of the binding of the AB to thetarget. Such linkers are generally referred to as flexible linkers.Suitable linkers can be readily selected and can be of any of a suitableof different lengths, such as from 1 amino acid (e.g., Gly) to 20 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 amino acids in length.

Exemplary flexible linkers include glycine polymers (G)n, glycine-serinepolymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 123) and(GGGS)n (SEQ ID NO: 124), where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers known in the art. Glycine and glycine-serine polymers arerelatively unstructured, and therefore may be able to serve as a neutraltether between components. Glycine accesses significantly more phi-psispace than even alanine, and is much less restricted than residues withlonger side chains (see Scheraga, Rev. Computational Chem. 11173-142(1992)). Exemplary flexible linkers include, but are not limited toGly-Gly-Ser-Gly (SEQ ID NO: 125), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 126),Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 127), Gly-Ser-Gly-Gly-Gly (SEQ ID NO:128), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 129), Gly-Ser-Ser-Ser-Gly (SEQ IDNO: 130), and the like. The ordinarily skilled artisan will recognizethat design of an activatable antibodies can include linkers that areall or partially flexible, such that the linker can include a flexiblelinker as well as one or more portions that confer less flexiblestructure to provide for a desired activatable antibodies structure.

In addition to the elements described above, the activatable antibodiescan contain additional elements such as, for example, amino acidsequence N- or C-terminal of the activatable antibodies. For example,activatable antibodies can include a targeting moiety to facilitatedelivery to a cell or tissue of interest. Activatable antibodies can beconjugated to an agent, such as a therapeutic agent, an antineoplasticagent, a toxin or fragment thereof, a detectable moiety or a diagnosticagent. Examples of agents are disclosed herein.

The activatable antibodies can also include any of the conjugatedagents, linkers and other components described herein in conjunctionwith an anti-Jagged antibody of the invention, including by way ofnon-limiting example, any of the agents listed in Table 30 and/or any ofthe linkers listed in Table 31 and/or Table 32.

Activatable Anti-Jagged Antibodies Having Non-Binding Steric Moieties orBinding Partners for Non-Binding Steric Moieties

The invention also provides activatable anti-Jagged antibodies thatinclude non-binding steric moieties (NB) or binding partners (BP) fornon-binding steric moieties, where the BP recruits or otherwise attractsthe NB to the activatable anti-Jagged antibody. The activatableanti-Jagged antibodies provided herein include, for example, anactivatable anti-Jagged antibody that includes a non-binding stericmoiety (NB), a cleavable linker (CL) and antibody or antibody fragment(AB) that binds Jagged 1 and Jagged 2; an activatable antibody thatincludes a binding partner for a non-binding steric moiety (BP), a CLand an AB; and an activatable anti-Jagged antibody that includes a BP towhich an NB has been recruited, a CL and an AB that binds Jagged 1 andJagged 2. Activatable antibodies in which the NB is covalently linked tothe CL and AB of the activatable anti-Jagged antibody or is associatedby interaction with a BP that is covalently linked to the CL and AB ofthe activatable anti-Jagged antibody are referred to herein as“NB-containing activatable anti-Jagged antibodies.” By activatable orswitchable is meant that the activatable antibody exhibits a first levelof binding to a target, i.e., Jagged 1 and/or Jagged 2, when theactivatable antibody is in an inhibited, masked or uncleaved state(i.e., a first conformation), and a second level of binding to thetarget when the activatable antibody is in an uninhibited, unmaskedand/or cleaved state (i.e., a second conformation, i.e., activatedantibody), where the second level of target binding is greater than thefirst level of target binding. The activatable antibody compositions canexhibit increased bioavailability and more favorable biodistributioncompared to conventional antibody therapeutics.

In some embodiments, activatable antibodies provide for reduced toxicityand/or adverse side effects that could otherwise result from binding ofthe anti-Jagged AB at non-treatment sites and/or non-diagnostic sites ifthe anti-Jagged AB were not masked or otherwise inhibited from bindingto such a site.

In one embodiment, the activatable antibody includes a non-bindingsteric moiety (NB); a cleavable linker (CL); and an antibody or antibodyfragment (AB) that binds specifically to Jagged 1 and Jagged 2, whereinthe NB is a polypeptide that does not bind specifically to the AB; theCL is a polypeptide that includes a substrate (S) for an enzyme; the CLis positioned such that in an uncleaved state, the NB interferes withbinding of the AB to Jagged 1 and/or Jagged 2 and in a cleaved state,the NB does not interfere with binding of the AB to Jagged 1 and/orJagged 2; and the NB does not inhibit cleavage of the CL by the enzyme.As used herein and throughout, the term polypeptide refers to anypolypeptide that includes at least two amino acid residues, includinglarger polypeptides, full-length proteins and fragments thereof, and theterm polypeptide is not limited to single-chain polypeptides and caninclude multi-unit, e.g., multi-chain, polypeptides. In cases where thepolypeptide is of a shorter length, for example, less than 50 aminoacids total, the terms peptide and polypeptide are used interchangeablyherein, and in cases where the polypeptide is of a longer length, forexample, 50 amino acids or greater, the terms polypeptide and proteinare used interchangeably herein.

In one embodiment, the activatable antibody includes a non-bindingsteric moiety (NB); a cleavable linker (CL); and an antibody or antibodyfragment (AB) that binds specifically to Jagged 1 and Jagged 2, wherein(i) the NB includes a polypeptide that does not bind specifically to theAB; (ii) CL is a polypeptide of up to 50 amino acids in length thatincludes a substrate (S) for an enzyme; (iii) the CL is positioned suchthat in an uncleaved state, the NB interferes with binding of the AB toJagged 1 and/or Jagged 2 and in a cleaved state, the NB does notinterfere with binding of the AB to Jagged 1 and/or Jagged 2; and (iv)the NB does not inhibit cleavage of the CL by the enzyme. For example,the CL has a length of up to 15 amino acids, a length of up to 20 aminoacids, a length of up to 25 amino acids, a length of up to 30 aminoacids, a length of up to 35 amino acids, a length of up to 40 aminoacids, a length of up to 45 amino acids, a length of up to 50 aminoacids, a length in the range of 10-50 amino acids, a length in the rangeof 15-50 amino acids, a length in the range of 20-50 amino acids, alength in the range of 25-50 amino acids, a length in the range of 30-50amino acids, a length in the range of 35-50 amino acids, a length in therange of 40-50 amino acids, a length in the range of 45-50 amino acids,a length in the range of 10-40 amino acids, a length in the range of15-40 amino acids, a length in the range of 20-40 amino acids, a lengthin the range of 25-40 amino acids, a length in the range of 30-40 aminoacids, a length in the range of 35-40 amino acids, a length in the rangeof 10-30 amino acids, a length in the range of 15-30 amino acids, alength in the range of 20-30 amino acids, a length in the range of 25-30amino acids, a length in the range of 10-20 amino acids, or a length inthe range of 10-15 amino acids.

In one embodiment, the activatable antibody includes a non-bindingsteric moiety (NB); a cleavable linker (CL); and an antibody or antibodyfragment (AB) that binds specifically to Jagged 1 and Jagged 2, wherein(i) the NB includes a polypeptide that does not bind specifically to theAB; (ii) the CL is a polypeptide that includes a substrate (S) for anenzyme; (iii) the CL is positioned such that in an uncleaved state, theNB interferes with binding of the AB to Jagged 1 and/or Jagged 2 and ina cleaved state, the NB does not interfere with binding of the AB toJagged 1 and/or Jagged 2; (iv) the NB does not inhibit cleavage of theCL by the enzyme; and (v) the activatable antibody has the structuralarrangement from N-terminus to C-terminus as follows in the uncleavedstate: NB-CL-AB or AB-CL-NB.

In one embodiment, the activatable antibody includes a non-bindingsteric moiety (NB); a cleavable linker (CL); and an antibody or antibodyfragment (AB) that binds specifically to Jagged 1 and Jagged 2, wherein(i) the NB includes a polypeptide that does not bind specifically to theAB; (ii) the CL is a polypeptide that includes a substrate (S) for anenzyme; (iii) the CL is positioned such that in an uncleaved state, theNB interferes with binding of the AB to Jagged 1 and/or Jagged 2 and ina cleaved state, the NB does not interfere with binding of the AB toJagged 1 and/or Jagged 2, and wherein the NB in the uncleavedactivatable antibody reduces the ability of the AB to bind Jagged 1and/or Jagged 2 by at least 50%, for example, by at least 60%, by atleast 70%, by at least 75%, by at least 80%, by at least 85%, by atleast 90%, by at least 95%, by at least 96%, by at least 97%, by atleast 98%, by at least 99%, by at least 100% as compared to the abilityof the cleaved AB to bind Jagged 1 and/or Jagged 2; and (iv) the NB doesnot inhibit cleavage of the CL by the enzyme. The reduction in theability of the AB to bind Jagged 1 and/or Jagged 2 is determined, forexample, using an assay as described herein or an in vitro targetdisplacement assay such as, for example, the assay described in PCTPublication Nos. WO 2009/025846 and WO 2010/081173.

In one embodiment, the activatable antibody includes a binding partner(BP) for a non-binding steric moiety (NB); a cleavable linker (CL); andan antibody or antibody fragment (AB) that binds specifically to Jagged1 and Jagged 2, wherein the BP is a polypeptide that binds to the NBwhen exposed thereto; the NB does not bind specifically to the AB; theCL is a polypeptide that includes a substrate (S) for an enzyme; the CLis positioned such that in an uncleaved state in the presence of the NB,the NB interferes with binding of the AB to Jagged 1 and/or Jagged 2 andin a cleaved state, the NB does not interfere with binding of the AB toJagged 1 and/or Jagged 2 and the BP does not interfere with binding ofthe AB to Jagged 1 and/or Jagged 2; and the NB and the BP do not inhibitcleavage of the CL by the enzyme. In some examples of this embodiment,the BP of the activatable antibody is optionally bound to the NB. In oneembodiment, the NB is recruited by the BP of the activatable antibody invivo.

In some examples of any of these activatable anti-Jagged antibodyembodiments, the activatable anti-Jagged antibody is formulated as acomposition. In some of these embodiments, the composition also includesthe NB, where the NB is co-formulated with the activatable anti-Jaggedantibody that includes the BP, the CL, and the AB. In some examples ofthis embodiment, the BP is selected from the group consisting of analbumin binding peptide, a fibrinogen binding peptide, a fibronectinbinding peptide, a hemoglobin binding peptide, a transferrin bindingpeptide, an immunoglobulin domain binding peptide, and other serumprotein binding peptides.

In some examples of any of these activatable anti-Jagged antibodyembodiments, the NB is a soluble, globular protein. In some examples ofany of these activatable anti-Jagged antibody embodiments, the NB is aprotein that circulates in the bloodstream. In some examples of any ofthese activatable anti-Jagged antibody embodiments, the NB is selectedfrom the group consisting of albumin, fibrinogen, fibronectin,hemoglobin, transferrin, an immunoglobulin domain, and other serumproteins.

In some examples of any of these activatable anti-Jagged antibodyembodiments, the CL is a polypeptide that includes a substrate (S) for aprotease. In some examples of any of these activatable anti-Jaggedantibody embodiments, the protease is co-localized with Jagged 1 and/orJagged 2 in a tissue, and the protease cleaves the CL in the activatableanti-Jagged antibody when the activatable antibody is exposed to theprotease. In some examples of any of these activatable anti-Jaggedantibody embodiments, the CL is a polypeptide of up to 50 amino acids inlength. In some examples of any of these activatable anti-Jaggedantibody embodiments, the CL is a polypeptide that includes a substrate(S) having a length of up to 15 amino acids, e.g., 3 amino acids long, 4amino acids long, 5 amino acids long, 6 amino acids long, 7 amino acidslong, 8 amino acids long, 9 amino acids long, 10 amino acids long, 11amino acids long, 12 amino acids long, 13 amino acids long, 14 aminoacids long, or 15 amino acids long.

In some examples of any of these activatable anti-Jagged antibodyembodiments, the activatable antibody has the structural arrangementfrom N-terminus to C-terminus as follows in the uncleaved state:NB-CL-AB, AB-CL-NB, BP-CL-AB or AB-CL-BP. In embodiments where theactivatable anti-Jagged antibody includes a BP and the activatableantibody is in the presence of the corresponding NB, the activatableantibody has a structural arrangement from N-terminus to C-terminus asfollows in the uncleaved state: NB:BP-CM-AB or AB-CM-BP:NB, where “:”represents an interaction, e.g., binding, between the NB and BP.

In some examples of any of these activatable anti-Jagged antibodyembodiments, the activatable antibody includes an antibody orantigen-binding fragment thereof that specifically binds Jagged 1 andJagged 2 and is a monoclonal antibody, domain antibody, single chain,Fab fragment, a F(ab)₂ fragment, a scFv, a scab, a dAb, a single domainheavy chain antibody, and a single domain light chain antibody. In someembodiments, such an antibody or immunologically active fragment thereofthat binds Jagged 1 and Jagged 2 is a mouse, chimeric, humanized orfully human monoclonal antibody.

In some examples of any of these activatable anti-Jagged antibodyembodiments, the activatable antibody includes a combination of a VHCDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1sequence, a VL CDR2 sequence, and a VL CDR3 sequence selected from thecombinations shown in Table 2. In some embodiments, the activatableantibody includes a combination of a VH CDR1 sequence, a VH CDR2sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence,and a VL CDR3 sequence that are at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the sequences shown in Table 2.

The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VHCDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence of at least one antibody selected from the group consisting ofthe 4D11 antibody, the 4B2 antibody, the 4E7 antibody, the 4E11antibody, the 6B7 antibody, and the 6F8 antibody.

The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VHCDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein at least one CDR sequence is selected from the groupconsisting of a VH CDR1 sequence that includes at least the amino acidsequence SYAMS (SEQ ID NO: 200); a VH CD2 sequence that includes atleast the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VHCDR3 sequence that includes at least the amino acid sequence DIGGRSAFDY(SEQ ID NO: 209); a VL CDR1 sequence that includes at least the aminoacid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes at least the amino acid sequence AASSLQS (SEQ ID NO: 211); anda VL CDR3 sequence that includes at least the amino acid sequenceQQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a combination of a VH CDR1 sequence, a VH CDR2 sequence, a VHCDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein at least one CDR sequence is selected from the groupconsisting of a VH CDR1 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence SYAMS (SEQ ID NO: 200); a VH CD2 sequencethat includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequenceSIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VH CDR3 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO:209); a VL CDR1 sequence that includes a sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQID NO: 211); and a VL CDR3 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence QQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a VH CDR1 sequence that includes at least the amino acidsequence SYAMS (SEQ ID NO: 200); a VH CD2 sequence that includes atleast the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VHCDR3 sequence that includes at least the amino acid sequence DIGGRSAFDY(SEQ ID NO: 209); a VL CDR1 sequence that includes at least the aminoacid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes at least the amino acid sequence AASSLQS (SEQ ID NO: 211); anda VL CDR3 sequence that includes at least the amino acid sequenceQQTVVAPPL (SEQ ID NO: 212).

The anti-Jagged antibodies of the invention include antibodies thatcontain a VH CDR1 sequence that includes a sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe amino acid sequence SYAMS (SEQ ID NO: 200); a VH CD2 sequence thatincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequenceSIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VH CDR3 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO:209); a VL CDR1 sequence that includes a sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence RASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence thatincludes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence AASSLQS (SEQID NO: 211); and a VL CDR3 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence QQTVVAPPL (SEQ ID NO: 212).

In some examples of any of these activatable anti-Jagged antibodyembodiments, the activatable antibody includes a combination of avariable heavy chain region and a variable light chain region selectedfrom the combinations listed in Table 4. In some embodiments, theactivatable antibody includes a combination of a variable heavy chainregion and a variable light chain region that are at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to thecombinations listed in Table 4.

In some examples of any of these activatable anti-Jagged antibodyembodiments, the activatable antibody also includes an agent conjugatedto the AB. In some embodiments, the agent is a therapeutic agent. Insome embodiments, the agent is an antineoplastic agent. In someembodiments, the agent is a toxin or fragment thereof. In someembodiments, the agent is conjugated to the AB via a linker. In someembodiments, the linker is a cleavable linker. In some embodiments, theagent is an agent selected from the group listed in Table 30. In someembodiments, the agent is a dolastatin. In some embodiments, the agentis an auristatin or derivative thereof. In some embodiments, the agentis auristatin E or a derivative thereof. In some embodiments, the agentis monomethyl auristatin E (MMAE). In some embodiments, the agent is amaytansinoid or maytansinoid derivative. In some embodiments, the agentis DM1 or DM4. In some embodiments, the agent is a duocarmycin orderivative thereof. In some embodiments, the agent is a calicheamicin orderivative thereof.

In some examples of any of these activatable anti-Jagged antibodyembodiments, the activatable antibody also includes a detectable moiety.In some embodiments, the detectable moiety is a diagnostic agent.

In some examples of any of these activatable anti-Jagged antibodyembodiments, the activatable antibody also includes a spacer. In someexamples of any of these activatable anti-Jagged antibody embodiments,the activatable antibody also includes a signal peptide. In someembodiments, the signal peptide is conjugated to the activatableantibody via a spacer. In some examples of any of these activatableanti-Jagged antibody embodiments, the spacer is joined directly to theMM of the activatable antibody.

In some examples of any of these activatable anti-Jagged antibodyembodiments, the serum half-life of the activatable antibody is at least5 days when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 4 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 3 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 2 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least24 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 20 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 18 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 16 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least14 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 12 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 10 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 8 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least6 hours when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 4 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 3 hours when administered to anorganism.

The invention also provides an isolated nucleic acid molecule encodingany of these activatable anti-Jagged antibodies, as well as vectors thatinclude these isolated nucleic acid sequences. The invention providesmethods of producing an activatable antibody by culturing a cell underconditions that lead to expression of the activatable antibody, whereinthe cell comprises such a nucleic acid sequence. In some embodiments,the cell comprises such a vector.

The dissociation constant (K_(d)) of the NB-containing activatableantibody toward the target is greater than the K_(d) of the AB towardsthe target when it is not associated with the NB or NB:BP. Thedissociation constant (K_(d)) of the NB-containing activatable antibodytoward the target is greater than the K_(d) of the parental AB towardsthe target. For example, the K_(d) of the NB-containing activatableantibody toward the target is at least 5, 10, 25, 50, 100, 250, 500,1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100,10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000,10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or100,000-10,000,000 times greater than the K_(d) of the AB when it is notassociated with the NB or NB:BP or the K_(d) of the parental AB towardsthe target. Conversely, the binding affinity of the NB-containingactivatable antibody towards the target is lower than the bindingaffinity of the AB when it is not associated with the NB or NB:BP orlower than the binding affinity of the parental AB towards the target.For example, the binding affinity of the NB-containing activatableantibody toward the target is at least 5, 10, 25, 50, 100, 250, 500,1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100,10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000,10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or100,000-10,000,000 times lower than the binding affinity of the AB whenit is not associated with the NB or NB:BP or lower than the bindingaffinity of the parental AB towards the target.

When the NB-containing activatable antibody is in the presence of Jagged1 and/or Jagged 2, specific binding of the AB to Jagged 1 and/or Jagged2 is reduced or inhibited, as compared to the specific binding of the ABwhen it is not associated with the NB or NB:BP. When the NB-containingactivatable antibody is in the presence of Jagged 1 and/or Jagged 2,specific binding of the AB to Jagged 1 and/or Jagged 2 is reduced orinhibited, as compared to the specific binding of the parental AB toJagged 1 and/or Jagged 2. When compared to the binding of the AB notassociated with an NB or NB:BP or the binding of the parental AB toJagged 1 and Jagged 2, the ability of the NB-containing activatableantibody to bind Jagged 1 and/or Jagged 2 is reduced, for example, by atleast 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72,84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measuredin vitro and/or in vivo.

When the NB-containing activatable antibody is in the presence of Jagged1 and Jagged 2 but not in the presence of a modifying agent (for examplea protease or other enzyme), specific binding of the AB to Jagged 1and/or Jagged 2 is reduced or inhibited, as compared to the specificbinding of the AB when it is not associated with the NB or NB:BP. Whenthe NB-containing activatable antibody is in the presence of Jagged 1and/or Jagged 2 but not in the presence of a modifying agent (forexample a protease, other enzyme, reduction agent, or light), specificbinding of the AB to Jagged 1 and/or Jagged 2 is reduced or inhibited,as compared to the specific binding of the parental AB to Jagged 1and/or Jagged 2. When compared to the binding of the AB not associatedwith an NB or NB:BP or the binding of the parental AB to Jagged 1 and/orJagged 2, the ability of the NB-containing activatable antibody to bindJagged 1 and/or Jagged 2 is reduced, for example, by at least 50%, 60%,70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% forat least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 months or longer when measured in vitro and/or invivo.

In some examples of any of these activatable antibody embodiments, theactivatable antibody includes an agent conjugated to the AB to producean activatable antibody conjugate. In some embodiments of theactivatable antibody conjugate, the agent is a therapeutic agent. Insome embodiments, the agent is a diagnostic agent. In some embodiments,the agent is a detectable marker. In some embodiments of the activatableantibody conjugate, the agent is an antineoplastic agent. In someembodiments of the activatable antibody conjugate, the agent is a toxinor fragment thereof. In some embodiments of the activatable antibodyconjugate, the agent is conjugated to the AB via a linker. In someembodiments of the activatable antibody conjugate, the linker is acleavable linker. In some embodiments, the agent is an agent selectedfrom the group listed in Table 30. In some embodiments, the agent is adolastatin. In some embodiments, the agent is an auristatin orderivative thereof. In some embodiments, the agent is auristatin E or aderivative thereof. In some embodiments, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is a maytansinoid ormaytansinoid derivative. In some embodiments, the agent is DM1 or DM4.In some embodiments, the agent is a duocarmycin or derivative thereof.In some embodiments, the agent is a calicheamicin or derivative thereof.

In some examples of any of these activatable antibody embodiments, theactivatable antibodies are dual-target binding activatable antibodies.Such dual target binding activatable antibodies contain two Abs that maybind the same or different targets. In specific embodiments,dual-targeting activatable antibodies contain bispecific antibodies orantibody fragments.

Dual target binding activatable antibodies are designed so as to have aCL cleavable by a cleaving agent that is co-localized in a target tissuewith one or both of the targets capable of binding to the ABs of theactivatable antibodies. Dual target binding activatable antibodies withmore than one AB to the same or different targets can be designed so asto have more than one CL, wherein the first CL is cleavable by acleaving agent in a first target tissue and wherein the second CL iscleavable by a cleaving agent in a second target tissue, with one ormore of the targets binding to the ABs of the activatable antibodies. Inone embodiment, the first and second target tissues are spatiallyseparated, for example, at different sites in the organism. In oneembodiment, the first and second target tissues are the same tissuetemporally separated, for example the same tissue at two differentpoints in time, for example the first time point is when the tissue isan early stage tumor, and the second time point is when the tissue is alate stage tumor.

The invention also provides nucleic acid molecules encoding theactivatable antibodies described herein. The invention also providesvectors that include these nucleic acids. The activatable antibodiesdescribed herein are produced by culturing a cell under conditions thatlead to expression of the activatable antibody, wherein the cellincludes these nucleic acid molecules or vectors.

The invention also provides methods of manufacturing activatableantibodies. In one embodiment, the method includes the steps of (a)culturing a cell that includes a nucleic acid construct that encodes theactivatable antibody under conditions that lead to expression of theactivatable antibody, wherein the activatable antibody includes (i) anon-binding steric moiety (NB); (ii) a cleavable linker (CL); and (iii)an antibody or an antigen binding fragment thereof (AB) thatspecifically binds a target, wherein (1) the NB does not bindspecifically to the AB; (2) the CL is a polypeptide that includes asubstrate (S) for an enzyme; (3) the CL is positioned such that in anuncleaved state, the NB interferes with binding of the AB to the targetand in a cleaved state, the NB does not interfere with binding of the ABto the target; and (4) the NB does not inhibit cleavage of the CL by theenzyme; and (b) recovering the activatable antibody.

In another embodiment, the method includes the steps of (a) culturing acell that includes a nucleic acid construct that encodes the activatableantibody under conditions that lead to expression of the activatableantibody, wherein the activatable antibody includes (i) a bindingpartner (BP) for a non-binding steric moiety (NB); (ii) a cleavablelinker (CL); and (iii) an antibody or an antigen binding fragmentthereof (AB) that specifically binds a target, wherein (1) the NB doesnot bind specifically to the AB; (2) the CL is a polypeptide thatincludes a substrate (S) for an enzyme; (3) the CL is positioned suchthat in an uncleaved state in the presence of the NB, the NB interfereswith binding of the AB to the target and in a cleaved state, the NB doesnot interfere with binding of the AB to the target and the BP does notinterfere with binding of the AB to the target; and (4) the NB and theBP do not inhibit cleavage of the CL by the enzyme; and (b) recoveringthe activatable antibody. In some examples of this embodiment, the BP ofthe activatable antibody is bound to the NB.

Use of Anti-Jagged Antibodies and Activatable Anti-Jagged Antibodies

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

In one embodiment, an antibody and/or an activatable antibody of theinvention, which include a monoclonal antibody of the invention (e.g., afully human monoclonal antibody) and/or an activatable antibody, may beused as therapeutic agents. Such agents will generally be employed todiagnose, prognose, monitor, treat, alleviate, and/or prevent a diseaseor pathology associated with Jagged 1 and/or Jagged 2 signaling throughNotch receptors in a subject. A therapeutic regimen is carried out byidentifying a subject, e.g., a human patient or other mammal sufferingfrom (or at risk of developing) a disorder such as a cancer, includingboth leukemias and solid tumors, or a fibrotic disorder, using standardmethods. An antibody and/or an activatable antibody preparation, forexample in some embodiments, one having high specificity and highaffinity for its target antigen, is administered to the subject and willgenerally have an effect due to its binding with the target.Administration of the antibody and/or an activatable antibody mayabrogate or inhibit or interfere with the signaling function of thetarget (e.g., Jagged 1 and/or Jagged 2 mediated signaling through Notchreceptors). Administration of the antibody and/or an activatableantibody may abrogate or inhibit or interfere with the binding of thetarget (e.g., Jagged 1 and/or Jagged 2) with an endogenous ligand (e.g.,a Notch receptor) to which it naturally binds. For example, the antibodyand/or an activatable antibody binds to the target and modulates,blocks, inhibits, reduces, antagonizes, neutralizes, or otherwiseinterferes with Jagged 1 and/or Jagged 2 mediated signaling throughNotch receptors.

Generally, alleviation or treatment of a disease or disorder involvesthe lessening of one or more symptoms or medical problems associatedwith the disease or disorder. For example, in the case of cancer, thetherapeutically effective amount of the drug can accomplish one or acombination of the following: reduce the number of cancer cells; reducethe tumor size; inhibit (i.e., to decrease to some extent and/or stop)cancer cell infiltration into peripheral organs; inhibit tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Insome embodiments, a composition of this invention can be used to preventthe onset or reoccurrence of the disease or disorder in a subject, e.g.,a human or other mammal, such as a non-human primate, companion animal(e.g., cat, dog, horse), farm animal, work animal, or zoo animal. Theterms subject and patient are used interchangeably herein

A therapeutically effective amount of an antibody and/or an activatableantibody of the invention relates generally to the amount needed toachieve a therapeutic objective. As noted above, this may be a bindinginteraction between the antibody and/or an activatable antibody and itstarget antigen that, in certain cases, interferes with the functioningof the target. The amount required to be administered will furthermoredepend on the binding affinity of the antibody and/or an activatableantibody for its specific antigen, and will also depend on the rate atwhich an administered antibody and/or an activatable antibody isdepleted from the free volume other subject to which it is administered.Common ranges for therapeutically effective dosing of an antibody and/orantibody fragment and/or an activatable antibody of the invention maybe, by way of nonlimiting example, from about 0.1 mg/kg body weight toabout 50 mg/kg body weight. Common dosing frequencies may range, forexample, from twice daily to once a week.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular inflammatory-relateddisorder. Alleviation of one or more symptoms of the cancer or fibroticdisorder indicates that the antibody and/or an activatable antibodyconfers a clinical benefit.

Methods for the screening of antibodies and/or activatable antibodiesthat possess the desired specificity include, but are not limited to,enzyme linked immunosorbent assay (ELISA) and other immunologicallymediated techniques known within the art.

In another embodiment, an antibody and/or an activatable antibodydirected against Jagged 1 and/or Jagged 2 are used in methods knownwithin the art relating to the localization and/or quantitation ofJagged 1 and/or Jagged 2 (e.g., for use in measuring levels of Jagged 1and/or Jagged 2 within appropriate physiological samples, for use indiagnostic methods, for use in imaging the protein, and the like). In agiven embodiment, an antibody and/or an activatable antibody specific toJagged 1 and/or Jagged 2, or a derivative, fragment, analog or homologthereof, that contain the antibody derived antigen binding domain, areutilized as pharmacologically active compounds (referred to hereinafteras “Therapeutics”).

In another embodiment, an antibody and/or an activatable antibodyspecific for Jagged 1 and/or Jagged 2 is used to isolate a Jagged 1and/or Jagged 2 polypeptide by standard techniques, such asimmunoaffinity, chromatography or immunoprecipitation. Antibodiesdirected against Jagged 1 and/or Jagged 2 and/or an activatable antibody(or a fragment thereof) are used diagnostically to monitor proteinlevels in tissue as part of a clinical testing procedure, e.g., todetermine the efficacy of a given treatment regimen. Detection can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

In yet another embodiment, an antibody and/or an activatable antibodyaccording to the invention can be used as an agent for detecting thepresence of Jagged 1 and/or Jagged 2 (or a fragment thereof) in asample. In some embodiments, the antibody contains a detectable label.Antibodies are polyclonal, or in some embodiments, monoclonal. An intactantibody, or a fragment thereof (e.g., F_(ab), scFv, or F_((ab)2)) isused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently-labeled secondary antibody and end-labeling of an antibodywith biotin such that it can be detected with fluorescently-labeledstreptavidin. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. Included within theusage of the term “biological sample”, therefore, is blood and afraction or component of blood including blood serum, blood plasma, orlymph. That is, the detection method of the invention can be used todetect a protein in a biological sample in vitro as well as in vivo. Forexample, in vitro techniques for detection of an analyte protein includeenzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. Procedures for conductingimmunoassays are described, for example in “ELISA: Theory and Practice:Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) HumanPress, Totowa, N.J., 1995; “Immunoassay”, E. Diamandis and T.Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and“Practice and Theory of Enzyme Immunoassays”, P. Tijssen, ElsevierScience Publishers, Amsterdam, 1985. Furthermore, in vivo techniques fordetection of an analyte protein include introducing into a subject alabeled anti-analyte protein antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

The anti-Jagged antibodies and/or activatable anti-Jagged antibodies ofthe invention are also useful in a variety of diagnostic andprophylactic formulations. In one embodiment, an anti-Jagged antibodyand/or activatable anti-Jagged antibody is administered to patients thatare at risk of developing one or more of the aforementioned cancer orfibrotic disorders. A patient's or organ's predisposition to one or moreof the aforementioned disorders can be determined using genotypic,serological or biochemical markers.

In another embodiment of the invention, an anti-Jagged antibody and/oractivatable anti-Jagged antibody is administered to human individualsdiagnosed with a clinical indication associated with one or more of theaforementioned disorders. Upon diagnosis, an anti-Jagged antibody and/oractivatable anti-Jagged antibody is administered to mitigate or reversethe effects of the clinical indication.

Antibodies and/or activatable antibodies of the invention are alsouseful in the detection of Jagged 1 and/or Jagged 2 in patient samplesand accordingly are useful as diagnostics. For example, the anti-Jaggedantibodies and/or activatable anti-Jagged antibodies of the inventionare used in in vitro assays, e.g., ELISA, to detect Jagged 1 and/orJagged 2 levels in a patient sample.

In one embodiment, an anti-Jagged antibody and/or activatableanti-Jagged antibody of the invention is immobilized on a solid support(e.g., the well(s) of a microtiter plate). The immobilized antibodyand/or activatable antibody serves as a capture antibody for any Jagged1 and/or Jagged 2 that may be present in a test sample. Prior tocontacting the immobilized antibody with a patient sample, the solidsupport is rinsed and treated with a blocking agent such as milk proteinor albumin to prevent nonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of Jagged antigen in the test sample is determined bycomparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained using theanti-Jagged antibodies of the invention in an in vitro diagnostic assay,it is possible to stage a disease in a subject based on expressionlevels of the Jagged antigen. For a given disease, samples of blood aretaken from subjects diagnosed as being at various stages in theprogression of the disease, and/or at various points in the therapeutictreatment of the disease. Using a population of samples that providesstatistically significant results for each stage of progression ortherapy, a range of concentrations of the antigen that may be consideredcharacteristic of each stage is designated.

Anti-Jagged antibodies and/or activatable anti-Jagged antibodies canalso be used in diagnostic and/or imaging methods. In some embodiments,such methods are in vitro methods. In some embodiments, such methods arein vivo methods. In some embodiments, such methods are in situ methods.In some embodiments, such methods are ex vivo methods. For example,activatable anti-Jagged antibodies having an enzymatically cleavable CMcan be used to detect the presence or absence of an enzyme that iscapable of cleaving the CM. Such activatable anti-Jagged antibodies canbe used in diagnostics, which can include in vivo detection (e.g.,qualitative or quantitative) of enzyme activity (or, in someembodiments, an environment of increased reduction potential such asthat which can provide for reduction of a disulfide bond) throughmeasured accumulation of activated anti-Jagged antibodies (i.e.,antibodies resulting from cleavage of an activatable anti-Jaggedantibody) in a given cell or tissue of a given host organism. Suchaccumulation of activated anti-Jagged antibodies indicates not only thatthe tissue expresses enzymatic activity (or an increased reductionpotential depending on the nature of the CM) but also that the tissueexpresses target to which the activated antibody binds.

For example, the CM can be selected to be a protease substrate for aprotease found at the site of a tumor, at the site of a viral orbacterial infection at a biologically confined site (e.g., such as in anabscess, in an organ, and the like), and the like. The AB can be onethat binds a target antigen. Using methods familiar to one skilled inthe art, a detectable label (e.g., a fluorescent label or radioactivelabel or radiotracer) can be conjugated to an AB or other region of ananti-Jagged antibody and/or activatable anti-Jagged antibody. Suitabledetectable labels are discussed in the context of the above screeningmethods and additional specific examples are provided below. Using an ABspecific to a protein or peptide of the disease state, along with aprotease whose activity is elevated in the disease tissue of interest,activatable anti-Jagged antibodies will exhibit an increased rate ofbinding to disease tissue relative to tissues where the CM specificenzyme is not present at a detectable level or is present at a lowerlevel than in disease tissue or is inactive (e.g., in zymogen form or incomplex with an inhibitor). Since small proteins and peptides arerapidly cleared from the blood by the renal filtration system, andbecause the enzyme specific for the CM is not present at a detectablelevel (or is present at lower levels in non-disease tissues or ispresent in inactive conformation), accumulation of activated anti-Jaggedantibodies in the disease tissue is enhanced relative to non-diseasetissues.

In another example, activatable anti-Jagged antibodies can be used todetect the presence or absence of a cleaving agent in a sample. Forexample, where the activatable anti-Jagged antibodies contain a CMsusceptible to cleavage by an enzyme, the activatable anti-Jaggedantibodies can be used to detect (either qualitatively orquantitatively) the presence of an enzyme in the sample. In anotherexample, where the activatable anti-Jagged antibodies contain a CMsusceptible to cleavage by reducing agent, the activatable anti-Jaggedantibodies can be used to detect (either qualitatively orquantitatively) the presence of reducing conditions in a sample. Tofacilitate analysis in these methods, the activatable antibodies can bedetectably labeled, and can be bound to a support (e.g., a solidsupport, such as a slide or bead). The detectable label can bepositioned on a portion of the activatable anti-Jagged antibody that isnot released following cleavage, for example, the detectable label canbe a quenched fluorescent label or other label that is not detectableuntil cleavage has occurred. The assay can be conducted by, for example,contacting the immobilized, detectably labeled activatable anti-Jaggedantibodies with a sample suspected of containing an enzyme and/orreducing agent for a time sufficient for cleavage to occur, then washingto remove excess sample and contaminants. The presence or absence of thecleaving agent (e.g., enzyme or reducing agent) in the sample is thenassessed by a change in detectable signal of the activatable anti-Jaggedantibodies prior to contacting with the sample e.g., the presence ofand/or an increase in detectable signal due to cleavage of theactivatable antibody by the cleaving agent in the sample.

Such detection methods can be adapted to also provide for detection ofthe presence or absence of a target that is capable of binding the AB ofthe activatable anti-Jagged antibodies when cleaved. Thus, the assayscan be adapted to assess the presence or absence of a cleaving agent andthe presence or absence of a target of interest. The presence or absenceof the cleaving agent can be detected by the presence of and/or anincrease in detectable label of the activatable anti-Jagged antibodiesas described above, and the presence or absence of the target can bedetected by detection of a target-AB complex e.g., by use of adetectably labeled anti-target antibody.

Activatable anti-Jagged antibodies are also useful in in situ imagingfor the validation of activatable antibody activation, e.g., by proteasecleavage, and binding to a particular target. In situ imaging is atechnique that enables localization of proteolytic activity and targetin biological samples such as cell cultures or tissue sections. Usingthis technique, it is possible to confirm both binding to a given targetand proteolytic activity based on the presence of a detectable label(e.g., a fluorescent label).

These techniques are useful with any frozen cells or tissue derived froma disease site (e.g. tumor tissue) or healthy tissues. These techniquesare also useful with fresh cell or tissue samples.

In these techniques, an activatable anti-Jagged antibody is labeled witha detectable label. The detectable label may be a fluorescent dye, (e.g.a fluorophore, Fluorescein Isothiocyanate (FITC), RhodamineIsothiocyanate (TRITC), an Alexa Fluor® label), a near infrared (NIR)dye (e.g., Qdot® nano crystals), a colloidal metal, a hapten, aradioactive marker, biotin and an amplification reagent such asstreptavidin, or an enzyme (e.g. horseradish peroxidase or alkalinephosphatase).

Detection of the label in a sample that has been incubated with thelabeled, activatable anti-Jagged antibody indicates that the samplecontains the target, i.e., Jagged 1 and/or Jagged 2, and contains aprotease that is specific for the CM of the activatable anti-Jaggedantibody. In some embodiments, the presence of the protease can beconfirmed using broad spectrum protease inhibitors such as thosedescribed herein, and/or by using an agent that is specific for theprotease, for example, an antibody such as A11, which is specific forthe protease matriptase (MT-SP1) and inhibits the proteolytic activityof MT-SP1; see e.g., International Publication Number WO 2010/129609,published 11 Nov. 2010. The same approach of using broad spectrumprotease inhibitors such as those described herein, and/or by using amore selective inhibitory agent can be used to identify a protease orclass of proteases specific for the CM of the activatable anti-Jaggedantibody. In some embodiments, the presence of the target can beconfirmed using an agent that is specific for the target, e.g., anotheranti-Jagged 1 and/or anti-Jagged 2 antibody, or the detectable label canbe competed with unlabeled Jagged 1 and/or Jagged 2. In someembodiments, unlabeled activatable anti-Jagged antibody could be used,with detection by a labeled secondary antibody or more complex detectionsystem.

Similar techniques are also useful for in vivo imaging where detectionof the fluorescent signal in a subject, e.g., a mammal, including ahuman, indicates that the disease site contains the target, i.e., Jagged1 and/or Jagged 2, and contains a protease that is specific for the CMof the activatable anti-Jagged antibody.

These techniques are also useful in kits and/or as reagents for thedetection, identification or characterization of protease activity in avariety of cells, tissues, and organisms based on the protease-specificCM in the activatable anti-Jagged antibody.

The invention provides methods of using the anti-Jagged antibodiesand/or activatable anti-Jagged antibodies in a variety of diagnosticand/or prophylactic indications. For example, the invention providesmethods of detecting presence or absence of a cleaving agent and atarget of interest in a subject or a sample by (i) contacting a subjector sample with an activatable anti-Jagged antibody, wherein theactivatable anti-Jagged antibody comprises a masking moiety (MM), acleavable moiety (CM) that is cleaved by the cleaving agent, and anantigen binding domain or fragment thereof (AB) that specifically bindsthe target of interest, wherein the activatable anti-Jagged antibody inan uncleaved, non-activated state comprises a structural arrangementfrom N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a)wherein the MM is a peptide that inhibits binding of the AB to theJagged target, and wherein the MM does not have an amino acid sequenceof a naturally occurring binding partner of the AB and is not a modifiedform of a natural binding partner of the AB; and (b) wherein, in anuncleaved, non-activated state, the MM interferes with specific bindingof the AB to the Jagged target, and in a cleaved, activated state the MMdoes not interfere or compete with specific binding of the AB to theJagged target; and (ii) measuring a level of activated activatableanti-Jagged antibody in the subject or sample, wherein a detectablelevel of activated activatable anti-Jagged antibody in the subject orsample indicates that the cleaving agent and a Jagged target are presentin the subject or sample and wherein no detectable level of activatedactivatable anti-Jagged antibody in the subject or sample indicates thatthe cleaving agent, a Jagged target or both the cleaving agent and aJagged target are absent and/or not sufficiently present in the subjector sample. In some embodiments, the activatable anti-Jagged antibody isan activatable anti-Jagged antibody to which a therapeutic agent isconjugated. In some embodiments, the activatable anti-Jagged antibody isnot conjugated to an agent. In some embodiments, the activatableanti-Jagged antibody comprises a detectable label. In some embodiments,the detectable label is positioned on the AB. In some embodiments,measuring the level of activatable anti-Jagged antibody in the subjector sample is accomplished using a secondary reagent that specificallybinds to the activated antibody, wherein the reagent comprises adetectable label. In some embodiments, the secondary reagent is anantibody comprising a detectable label.

The invention also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orsample with an activatable anti-Jagged antibody in the presence of aJagged target of interest, e.g., Jagged 1 and/or Jagged 2, wherein theactivatable anti-Jagged antibody comprises a masking moiety (MM), acleavable moiety (CM) that is cleaved by the cleaving agent, and anantigen binding domain or fragment thereof (AB) that specifically bindsthe target of interest, wherein the activatable anti-Jagged antibody inan uncleaved, non-activated state comprises a structural arrangementfrom N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a)wherein the MM is a peptide that inhibits binding of the AB to theJagged target, and wherein the MM does not have an amino acid sequenceof a naturally occurring binding partner of the AB and is not a modifiedform of a natural binding partner of the AB; and (b) wherein, in anuncleaved, non-activated state, the MM interferes with specific bindingof the AB to the Jagged target, and in a cleaved, activated state the MMdoes not interfere or compete with specific binding of the AB to theJagged target; and (ii) measuring a level of activated activatableanti-Jagged antibody in the subject or sample, wherein a detectablelevel of activated activatable anti-Jagged antibody in the subject orsample indicates that the cleaving agent is present in the subject orsample and wherein no detectable level of activated activatableanti-Jagged antibody in the subject or sample indicates that thecleaving agent is absent and/or not sufficiently present in the subjector sample. In some embodiments, the activatable anti-Jagged antibody isan activatable anti-Jagged antibody to which a therapeutic agent isconjugated. In some embodiments, the activatable anti-Jagged antibody isnot conjugated to an agent. In some embodiments, the activatableanti-Jagged antibody comprises a detectable label. In some embodiments,the detectable label is positioned on the AB. In some embodiments,measuring the level of activatable anti-Jagged antibody in the subjector sample is accomplished using a secondary reagent that specificallybinds to the activated antibody, wherein the reagent comprises adetectable label. In some embodiments, the secondary reagent is anantibody comprising a detectable label.

The invention also provides kits for use in methods of detectingpresence or absence of a cleaving agent and a Jagged target of interest(e.g., Jagged 1 and/or Jagged 2) in a subject or a sample, where thekits include at least an activatable anti-Jagged antibody comprises amasking moiety (MM), a cleavable moiety (CM) that is cleaved by thecleaving agent, and an antigen binding domain or fragment thereof (AB)that specifically binds the target of interest, wherein the activatableanti-Jagged antibody in an uncleaved, non-activated state comprises astructural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that inhibitsbinding of the AB to the Jagged target, and wherein the MM does not havean amino acid sequence of a naturally occurring binding partner of theAB and is not a modified form of a natural binding partner of the AB;and (b) wherein, in an uncleaved, non-activated state, the MM interfereswith specific binding of the AB to the Jagged target, and in a cleaved,activated state the MM does not interfere or compete with specificbinding of the AB to the Jagged target; and (ii) measuring a level ofactivated activatable anti-Jagged antibody in the subject or sample,wherein a detectable level of activated activatable anti-Jagged antibodyin the subject or sample indicates that the cleaving agent is present inthe subject or sample and wherein no detectable level of activatedactivatable anti-Jagged antibody in the subject or sample indicates thatthe cleaving agent is absent and/or not sufficiently present in thesubject or sample. In some embodiments, the activatable anti-Jaggedantibody is an activatable anti-Jagged antibody to which a therapeuticagent is conjugated. In some embodiments, the activatable anti-Jaggedantibody is not conjugated to an agent. In some embodiments, theactivatable anti-Jagged antibody comprises a detectable label. In someembodiments, the detectable label is positioned on the AB. In someembodiments, measuring the level of activatable anti-Jagged antibody inthe subject or sample is accomplished using a secondary reagent thatspecifically binds to the activated antibody, wherein the reagentcomprises a detectable label. In some embodiments, the secondary reagentis an antibody comprising a detectable label.

The invention also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orsample with an activatable anti-Jagged antibody, wherein the activatableanti-Jagged antibody comprises a masking moiety (MM), a cleavable moiety(CM) that is cleaved by the cleaving agent, an antigen binding domain(AB) that specifically binds a Jagged target, e.g., Jagged 1 and/orJagged 2, and a detectable label, wherein the activatable anti-Jaggedantibody in an uncleaved, non-activated state comprises a structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM; wherein the MM is a peptide that inhibits binding of the AB tothe Jagged target, and wherein the MM does not have an amino acidsequence of a naturally occurring binding partner of the AB and is not amodified form of a natural binding partner of the AB; wherein, in anuncleaved, non-activated state, the MM interferes with specific bindingof the AB to the Jagged target, and in a cleaved, activated state the MMdoes not interfere or compete with specific binding of the AB to theJagged target; and wherein the detectable label is positioned on aportion of the activatable anti-Jagged antibody that is releasedfollowing cleavage of the CM; and (ii) measuring a level of detectablelabel in the subject or sample, wherein a detectable level of thedetectable label in the subject or sample indicates that the cleavingagent is absent and/or not sufficiently present in the subject or sampleand wherein no detectable level of the detectable label in the subjector sample indicates that the cleaving agent is present in the subject orsample. In some embodiments, the activatable anti-Jagged antibody is anactivatable anti-Jagged antibody to which a therapeutic agent isconjugated. In some embodiments, the activatable anti-Jagged antibody isnot conjugated to an agent. In some embodiments, the activatableanti-Jagged antibody comprises a detectable label. In some embodiments,the detectable label is positioned on the AB. In some embodiments,measuring the level of activatable anti-Jagged antibody in the subjector sample is accomplished using a secondary reagent that specificallybinds to the activated antibody, wherein the reagent comprises adetectable label. In some embodiments, the secondary reagent is anantibody comprising a detectable label.

The invention also provides kits for use in methods of detectingpresence or absence of a cleaving agent and a Jagged target of interest(e.g., Jagged 1 and/or Jagged 2) in a subject or a sample, where thekits include at least an activatable anti-Jagged antibody and/orconjugated activatable anti-Jagged antibody (e.g., an activatableantibody to which a therapeutic agent is conjugated) described hereinfor use in contacting a subject or biological sample and means fordetecting the level of activated activatable anti-Jagged antibody and/orconjugated activatable anti-Jagged antibody in the subject or biologicalsample, wherein a detectable level of activated activatable anti-Jaggedantibody in the subject or biological sample indicates that the cleavingagent and the Jagged target are present in the subject or biologicalsample and wherein no detectable level of activated activatableanti-Jagged antibody in the subject or biological sample indicates thatthe cleaving agent, the Jagged target or both the cleaving agent and theJagged target are absent and/or not sufficiently present in the subjector biological sample, such that Jagged target binding and/or proteasecleavage of the activatable anti-Jagged antibody cannot be detected inthe subject or biological sample.

The invention also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orbiological sample with an activatable anti-Jagged antibody in thepresence of the Jagged target, and (ii) measuring a level of activatedactivatable anti-Jagged antibody in the subject or biological sample,wherein a detectable level of activated activatable anti-Jagged antibodyin the subject or biological sample indicates that the cleaving agent ispresent in the subject or biological sample and wherein no detectablelevel of activated activatable anti-Jagged antibody in the subject orbiological sample indicates that the cleaving agent is absent and/or notsufficiently present in the subject or biological sample at a detectablelevel, such that protease cleavage of the activatable anti-Jaggedantibody cannot be detected in the subject or biological sample. Such anactivatable anti-Jagged antibody includes a masking moiety (MM), acleavable moiety (CM) that is cleaved by the cleaving agent, and anantigen binding domain or fragment thereof (AB) that specifically bindsthe Jagged target, wherein the activatable anti-Jagged antibody in anuncleaved (i.e., non-activated) state comprises a structural arrangementfrom N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a)wherein the MM is a peptide that inhibits binding of the AB to theJagged target, and wherein the MM does not have an amino acid sequenceof a naturally occurring binding partner of the AB; and (b) wherein theMM of the activatable anti-Jagged antibody in an uncleaved stateinterferes with specific binding of the AB to the Jagged target, andwherein the MM of an activatable anti-Jagged antibody in a cleaved(i.e., activated) state does not interfere or compete with specificbinding of the AB to the Jagged target. In some embodiments, theactivatable anti-Jagged antibody is an activatable anti-Jagged antibodyto which a therapeutic agent is conjugated. In some embodiments, theactivatable anti-Jagged antibody is not conjugated to an agent. In someembodiments, the detectable label is attached to the masking moiety. Insome embodiments, the detectable label is attached to the cleavablemoiety N-terminal to the protease cleavage site. In some embodiments, asingle antigen binding site of the AB is masked. In some embodimentswherein an antibody of the disclosure has at least two antigen bindingsites, at least one antigen binding site is masked and at least oneantigen binding site is not masked. In some embodiments all antigenbinding sites are masked. In some embodiments, the measuring stepincludes use of a secondary reagent comprising a detectable label.

The invention also provides kits for use in methods of detectingpresence or absence of a cleaving agent and a Jagged target of interest(e.g., Jagged 1 and/or Jagged 2) in a subject or a sample, where thekits include at least an activatable anti-Jagged antibody and/orconjugated activatable anti-Jagged antibody described herein for use incontacting a subject or biological sample with an activatableanti-Jagged antibody in the presence of the Jagged target, and measuringa level of activated activatable anti-Jagged antibody in the subject orbiological sample, wherein a detectable level of activated activatableanti-Jagged antibody in the subject or biological sample indicates thatthe cleaving agent is present in the subject or biological sample andwherein no detectable level of activated activatable anti-Jaggedantibody in the subject or biological sample indicates that the cleavingagent is absent and/or not sufficiently present in the subject orbiological sample at a detectable level, such that protease cleavage ofthe activatable anti-Jagged antibody cannot be detected in the subjector biological sample. Such an activatable anti-Jagged antibody includesa masking moiety (MM), a cleavable moiety (CM) that is cleaved by thecleaving agent, and an antigen binding domain or fragment thereof (AB)that specifically binds the Jagged target, wherein the activatableanti-Jagged antibody in an uncleaved (i.e., non-activated) statecomprises a structural arrangement from N-terminus to C-terminus asfollows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide thatinhibits binding of the AB to the Jagged target, and wherein the MM doesnot have an amino acid sequence of a naturally occurring binding partnerof the AB; and (b) wherein the MM of the activatable anti-Jaggedantibody in an uncleaved state interferes with specific binding of theAB to the Jagged target, and wherein the MM of an activatableanti-Jagged antibody in a cleaved (i.e., activated) state does notinterfere or compete with specific binding of the AB to the Jaggedtarget. In some embodiments, the activatable anti-Jagged antibody is anactivatable anti-Jagged antibody to which a therapeutic agent isconjugated. In some embodiments, the activatable anti-Jagged antibody isnot conjugated to an agent. In some embodiments, the detectable label isattached to the masking moiety. In some embodiments, the detectablelabel is attached to the cleavable moiety N-terminal to the proteasecleavage site. In some embodiments, a single antigen binding site of theAB is masked. In some embodiments wherein an antibody of the disclosurehas at least two antigen binding sites, at least one antigen bindingsite is masked and at least one antigen binding site is not masked. Insome embodiments all antigen binding sites are masked. In someembodiments, the measuring step includes use of a secondary reagentcomprising a detectable label.

The invention also provides kits for use in methods of detectingpresence or absence of a cleaving agent in a subject or a sample, wherethe kits include at least an activatable anti-Jagged antibody and/orconjugated activatable anti-Jagged antibody described herein for use incontacting a subject or biological sample and means for detecting thelevel of activated activatable anti-Jagged antibody and/or conjugatedactivatable anti-Jagged antibody in the subject or biological sample,wherein the activatable anti-Jagged antibody includes a detectable labelthat is positioned on a portion of the activatable anti-Jagged antibodythat is released following cleavage of the CM, wherein a detectablelevel of activated activatable anti-Jagged antibody in the subject orbiological sample indicates that the cleaving agent is absent and/or notsufficiently present in the subject or biological sample such thatJagged target binding and/or protease cleavage of the activatableanti-Jagged antibody cannot be detected in the subject or biologicalsample, and wherein no detectable level of activated activatableanti-Jagged antibody in the subject or biological sample indicates thatthe cleaving agent is present in the subject or biological sample at adetectable level.

The invention provides methods of detecting presence or absence of acleaving agent and the Jagged target in a subject or a sample by (i)contacting a subject or biological sample with an activatableanti-Jagged antibody, wherein the activatable anti-Jagged antibodyincludes a detectable label that is positioned on a portion of theactivatable anti-Jagged antibody that is released following cleavage ofthe CM and (ii) measuring a level of activated activatable anti-Jaggedantibody in the subject or biological sample, wherein a detectable levelof activated activatable anti-Jagged antibody in the subject orbiological sample indicates that the cleaving agent, the Jagged targetor both the cleaving agent and the Jagged target are absent and/or notsufficiently present in the subject or biological sample, such thatJagged target binding and/or protease cleavage of the activatableanti-Jagged antibody cannot be detected in the subject or biologicalsample, and wherein a reduced detectable level of activated activatableanti-Jagged antibody in the subject or biological sample indicates thatthe cleaving agent and the Jagged target are present in the subject orbiological sample. A reduced level of detectable label is, for example,a reduction of about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95% and/or about 100%. Such an activatable anti-Jagged antibodyincludes a masking moiety (MM), a cleavable moiety (CM) that is cleavedby the cleaving agent, and an antigen binding domain or fragment thereof(AB) that specifically binds the Jagged target, wherein the activatableanti-Jagged antibody in an uncleaved (i.e., non-activated) statecomprises a structural arrangement from N-terminus to C-terminus asfollows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide thatinhibits binding of the AB to the Jagged target, and wherein the MM doesnot have an amino acid sequence of a naturally occurring binding partnerof the AB; and (b) wherein the MM of the activatable anti-Jaggedantibody in an uncleaved state interferes with specific binding of theAB to the Jagged target, and wherein the MM of an activatableanti-Jagged antibody in a cleaved (i.e., activated) state does notinterfere or compete with specific binding of the AB to the Jaggedtarget. In some embodiments, the activatable anti-Jagged antibody is anactivatable anti-Jagged antibody to which a therapeutic agent isconjugated. In some embodiments, the activatable anti-Jagged antibody isnot conjugated to an agent. In some embodiments, the activatableanti-Jagged antibody comprises a detectable label. In some embodiments,the detectable label is positioned on the AB. In some embodiments,measuring the level of activatable anti-Jagged antibody in the subjector sample is accomplished using a secondary reagent that specificallybinds to the activated antibody, wherein the reagent comprises adetectable label. In some embodiments, the secondary reagent is anantibody comprising a detectable label.

The invention also provides kits for use in methods of detectingpresence or absence of a cleaving agent and a Jagged target of interestin a subject or a sample, where the kits include at least an activatableanti-Jagged antibody and/or conjugated activatable anti-Jagged antibodydescribed herein for use in contacting a subject or biological sampleand means for detecting the level of activated activatable anti-Jaggedantibody and/or conjugated activatable anti-Jagged antibody in thesubject or biological sample, wherein a detectable level of activatedactivatable anti-Jagged antibody in the subject or biological sampleindicates that the cleaving agent, the Jagged target or both thecleaving agent and the Jagged target are absent and/or not sufficientlypresent in the subject or biological sample, such that Jagged targetbinding and/or protease cleavage of the activatable anti-Jagged antibodycannot be detected in the subject or biological sample, and wherein areduced detectable level of activated activatable anti-Jagged antibodyin the subject or biological sample indicates that the cleaving agentand the Jagged target are present in the subject or biological sample. Areduced level of detectable label is, for example, a reduction of about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about100%.

The invention also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orbiological sample with an activatable anti-Jagged antibody, wherein theactivatable anti-Jagged antibody includes a detectable label that ispositioned on a portion of the activatable anti-Jagged antibody that isreleased following cleavage of the CM; and (ii) measuring a level ofdetectable label in the subject or biological sample, wherein adetectable level of the detectable label in the subject or biologicalsample indicates that the cleaving agent is absent and/or notsufficiently present in the subject or biological sample at a detectablelevel, such that protease cleavage of the activatable anti-Jaggedantibody cannot be detected in the subject or biological sample, andwherein a reduced detectable level of the detectable label in thesubject or biological sample indicates that the cleaving agent ispresent in the subject or biological sample. A reduced level ofdetectable label is, for example, a reduction of about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95% and/or about 100%. Such anactivatable anti-Jagged antibody includes a masking moiety (MM), acleavable moiety (CM) that is cleaved by the cleaving agent, and anantigen binding domain or fragment thereof (AB) that specifically bindsthe Jagged target, wherein the activatable anti-Jagged antibody in anuncleaved (i.e., non-activated) state comprises a structural arrangementfrom N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a)wherein the MM is a peptide that inhibits binding of the AB to theJagged target, and wherein the MM does not have an amino acid sequenceof a naturally occurring binding partner of the AB; and (b) wherein theMM of the activatable anti-Jagged antibody in an uncleaved stateinterferes with specific binding of the AB to the Jagged target, andwherein the MM of an activatable anti-Jagged antibody in a cleaved(i.e., activated) state does not interfere or compete with specificbinding of the AB to the Jagged target. In some embodiments, theactivatable anti-Jagged antibody is an activatable anti-Jagged antibodyto which a therapeutic agent is conjugated. In some embodiments, theactivatable anti-Jagged antibody is not conjugated to an agent. In someembodiments, the activatable anti-Jagged antibody comprises a detectablelabel. In some embodiments, the detectable label is positioned on theAB. In some embodiments, measuring the level of activatable anti-Jaggedantibody in the subject or sample is accomplished using a secondaryreagent that specifically binds to the activated antibody, wherein thereagent comprises a detectable label. In some embodiments, the secondaryreagent is an antibody comprising a detectable label.

The invention also provides kits for use in methods of detectingpresence or absence of a cleaving agent of interest in a subject or asample, where the kits include at least an activatable anti-Jaggedantibody and/or conjugated activatable anti-Jagged antibody describedherein for use in contacting a subject or biological sample and meansfor detecting the level of activated activatable anti-Jagged antibodyand/or conjugated activatable anti-Jagged antibody in the subject orbiological sample, wherein the activatable anti-Jagged antibody includesa detectable label that is positioned on a portion of the activatableanti-Jagged antibody that is released following cleavage of the CM,wherein a detectable level of the detectable label in the subject orbiological sample indicates that the cleaving agent, the Jagged target,or both the cleaving agent and the Jagged target are absent and/or notsufficiently present in the subject or biological sample, such thatJagged target binding and/or protease cleavage of the activatableanti-Jagged antibody cannot be detected in the subject or biologicalsample, and wherein a reduced detectable level of the detectable labelin the subject or biological sample indicates that the cleaving agentand the Jagged target are present in the subject or biological sample. Areduced level of detectable label is, for example, a reduction of about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or about100%.

In some embodiments of these methods and kits, the activatableanti-Jagged antibody includes a detectable label. In some embodiments ofthese methods and kits, the detectable label includes an imaging agent,a contrasting agent, an enzyme, a fluorescent label, a chromophore, adye, one or more metal ions, or a ligand-based label. In someembodiments of these methods and kits, the imaging agent comprises aradioisotope. In some embodiments of these methods and kits, theradioisotope is indium or technetium. In some embodiments of thesemethods and kits, the contrasting agent comprises iodine, gadolinium oriron oxide. In some embodiments of these methods and kits, the enzymecomprises horseradish peroxidase, alkaline phosphatase, orβ-galactosidase. In some embodiments of these methods and kits, thefluorescent label comprises yellow fluorescent protein (YFP), cyanfluorescent protein (CFP), green fluorescent protein (GFP), modified redfluorescent protein (mRFP), red fluorescent protein tdimer2 (RFPtdimer2), HCRED, or a europium derivative. In some embodiments of thesemethods and kits, the luminescent label comprises an N-methylacrydiumderivative. In some embodiments of these methods, the label comprises anAlexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In someembodiments of these methods and kits, the ligand-based label comprisesbiotin, avidin, streptavidin or one or more haptens.

In some embodiments of these methods and kits, the subject is a mammal.In some embodiments of these methods and kits, the subject is a human.In some embodiments, the subject is a non-human mammal, such as anon-human primate, companion animal (e.g., cat, dog, horse), farmanimal, work animal, or zoo animal. In some embodiments, the subject isa rodent.

In some embodiments of these methods, the method is an in vivo method.In some embodiments of these methods, the method is an in situ method.In some embodiments of these methods, the method is an ex vivo method.In some embodiments of these methods, the method is an in vitro method.

In some embodiments, in situ imaging and/or in vivo imaging are usefulin methods to identify which patients to treat. For example, in in situimaging, the activatable anti-Jagged antibodies are used to screenpatient samples to identify those patients having the appropriateprotease(s) and target(s) at the appropriate location, e.g., at a tumorsite.

In some embodiments in situ imaging is used to identify or otherwiserefine a patient population suitable for treatment with an activatableanti-Jagged antibody of the disclosure. For example, patients that testpositive for both the target (e.g., Jagged 1 and/or Jagged 2) and aprotease that cleaves the substrate in the cleavable moiety (CM) of theactivatable anti-Jagged antibody being tested (e.g., accumulateactivated antibodies at the disease site) are identified as suitablecandidates for treatment with such an activatable anti-Jagged antibodycomprising such a CM. Likewise, patients that test negative for eitheror both of the target (e.g., Jagged 1 and/or Jagged 2) and the proteasethat cleaves the substrate in the CM in the activatable antibody beingtested using these methods might be identified as suitable candidatesfor another form of therapy. In some embodiments, such patients thattest negative with respect to a first activatable anti-Jagged antibodycan be tested with other activatable anti-Jagged antibodies comprisingdifferent CMs until a suitable activatable anti-Jagged antibody fortreatment is identified (e.g., an activatable anti-Jagged antibodycomprising a CM that is cleaved by the patient at the site of disease).

In some embodiments in vivo imaging is used to identify or otherwiserefine a patient population suitable for treatment with an activatableanti-Jagged antibody of the disclosure. For example, patients that testpositive for both the target (e.g., Jagged 1 and/or Jagged 2) and aprotease that cleaves the substrate in the cleavable moiety (CM) of theactivatable anti-Jagged antibody being tested (e.g., accumulateactivated antibodies at the disease site) are identified as suitablecandidates for treatment with such an activatable anti-Jagged antibodycomprising such a CM. Likewise, patients that test negative might beidentified as suitable candidates for another form of therapy. In someembodiments, such patients that test negative with respect to a firstactivatable anti-Jagged antibody can be tested with other activatableanti-Jagged antibodies comprising different CMs until a suitableactivatable anti-Jagged antibody for treatment is identified (e.g., anactivatable anti-Jagged antibody comprising a CM that is cleaved by thepatient at the site of disease).

In some embodiments of the methods and kits, the method or kit is usedto identify or otherwise refine a patient population suitable fortreatment with an activatable anti-Jagged antibody of the disclosure.For example, patients that test positive for both the target (e.g.,Jagged 1 and/or Jagged 2) and a protease that cleaves the substrate inthe cleavable moiety (CM) of the activatable anti-Jagged antibody beingtested in these methods are identified as suitable candidates fortreatment with such an activatable anti-Jagged antibody comprising sucha CM. Likewise, patients that test negative for both of the targets(e.g., Jagged 1 and Jagged 2) and the protease that cleaves thesubstrate in the CM in the activatable antibody being tested using thesemethods might be identified as suitable candidates for another form oftherapy. In some embodiments, such patients can be tested with otheractivatable anti-Jagged antibodies until a suitable activatableanti-Jagged antibody for treatment is identified (e.g., an activatableanti-Jagged antibody comprising a CM that is cleaved by the patient atthe site of disease). In some embodiments, patients that test negativefor either of the targets (e.g., Jagged 1 or Jagged 2) are identified assuitable candidates for treatment with such an activatable anti-Jaggedantibody comprising such a CM. In some embodiments, patients that testnegative for either of the targets (e.g., Jagged 1 or Jagged 2) areidentified as not being suitable candidates for treatment with such anactivatable anti-Jagged antibody comprising such a CM. In someembodiments, such patients can be tested with other activatableanti-Jagged antibodies until a suitable activatable anti-Jagged antibodyfor treatment is identified (e.g., an activatable anti-Jagged antibodycomprising a CM that is cleaved by the patient at the site of disease).In some embodiments, the activatable anti-Jagged antibody is anactivatable anti-Jagged antibody to which a therapeutic agent isconjugated. In some embodiments, the activatable anti-Jagged antibody isnot conjugated to an agent. In some embodiments, the activatableanti-Jagged antibody comprises a detectable label. In some embodiments,the detectable label is positioned on the AB. In some embodiments,measuring the level of activatable anti-Jagged antibody in the subjector sample is accomplished using a secondary reagent that specificallybinds to the activated antibody, wherein the reagent comprises adetectable label. In some embodiments, the secondary reagent is anantibody comprising a detectable label.

In some embodiments, a method or kit is used to identify or otherwiserefine a patient population suitable for treatment with an anti-Jaggedactivatable antibody and/or conjugated activatable anti-Jagged antibody(e.g., activatable antibody to which a therapeutic agent is conjugated)of the disclosure, followed by treatment by administering thatactivatable anti-Jagged antibody and/or conjugated activatableanti-Jagged antibody to a subject in need thereof. For example, patientsthat test positive for both the targets (e.g., Jagged 1 and Jagged 2)and a protease that cleaves the substrate in the cleavable moiety (CM)of the activatable anti-Jagged antibody and/or conjugated activatableanti-Jagged antibody being tested in these methods are identified assuitable candidates for treatment with such antibody and/or such aconjugated activatable anti-Jagged antibody comprising such a CM, andthe patient is then administered a therapeutically effective amount ofthe activatable anti-Jagged antibody and/or conjugated activatableanti-Jagged antibody that was tested. Likewise, patients that testnegative for either or both of the target (e.g., Jagged 1 and/or Jagged2) and the protease that cleaves the substrate in the CM in theactivatable anti-Jagged antibody being tested using these methods mightbe identified as suitable candidates for another form of therapy. Insome embodiments, such patients can be tested with other antibody and/orconjugated activatable anti-Jagged antibody until a suitable antibodyand/or conjugated activatable anti-Jagged antibody for treatment isidentified (e.g., an activatable anti-Jagged antibody and/or conjugatedactivatable anti-Jagged antibody comprising a CM that is cleaved by thepatient at the site of disease). In some embodiments, the patient isthen administered a therapeutically effective amount of the activatableanti-Jagged antibody and/or conjugated for which the patient testedpositive.

In some embodiments of these methods and kits, the MM is a peptidehaving a length from about 4 to 40 amino acids. In some embodiments ofthese methods and kits, the activatable anti-Jagged antibody comprises alinker peptide, wherein the linker peptide is positioned between the MMand the CM. In some embodiments of these methods and kits, theactivatable anti-Jagged antibody comprises a linker peptide, where thelinker peptide is positioned between the AB and the CM. In someembodiments of these methods and kits, the activatable anti-Jaggedantibody comprises a first linker peptide (L1) and a second linkerpeptide (L2), wherein the first linker peptide is positioned between theMM and the CM and the second linker peptide is positioned between the ABand the CM. In some embodiments of these methods and kits, each of L1and L2 is a peptide of about 1 to 20 amino acids in length, and whereineach of L1 and L2 need not be the same linker. In some embodiments ofthese methods and kits, one or both of L1 and L2 comprises aglycine-serine polymer. In some embodiments of these methods and kits,at least one of L1 and L2 comprises an amino acid sequence selected fromthe group consisting of (GS)n, (GSGGS)n (SEQ ID NO: 123) and (GGGS)n(SEQ ID NO: 124), where n is an integer of at least one. In someembodiments of these methods and kits, at least one of L1 and L2comprises an amino acid sequence having the formula (GGS)n, where n isan integer of at least one. In some embodiments of these methods andkits, at least one of L1 and L2 comprises an amino acid sequenceselected from the group consisting of Gly-Gly-Ser-Gly (SEQ ID NO: 125),Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 126), Gly-Ser-Gly-Ser-Gly (SEQ ID NO:127), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 128), Gly-Gly-Gly-Ser-Gly (SEQ IDNO: 129), and Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 130).

In some embodiments of these methods and kits, the AB comprises anantibody or antibody fragment sequence selected from the cross-reactiveanti-Jagged antibody sequences presented herein. In some embodiments ofthese methods and kits, the AB comprises a Fab fragment, a scFv or asingle chain antibody (scAb).

In some embodiments of these methods and kits, the cleaving agent is aprotease that is co-localized in the subject or sample with the Jaggedtarget and the CM is a polypeptide that functions as a substrate for theprotease, wherein the protease cleaves the CM in the activatableanti-Jagged antibody when the activatable anti-Jagged antibody isexposed to the protease. In some embodiments of these methods and kits,the CM is a polypeptide of up to 15 amino acids in length. In someembodiments of these methods and kits, the CM is coupled to theN-terminus of the AB. In some embodiments of these methods and kits, theCM is coupled to the C-terminus of the AB. In some embodiments of thesemethods and kits, the CM is coupled to the N-terminus of a VL chain ofthe AB.

In some embodiments of these methods and kits, the cleaving agent is anenzyme and the CM is a substrate for the enzyme. In some embodiments ofthese methods and kits, the enzyme is a protease disclosed herein. Insome embodiments of these methods and kits, the protease is one of theproteases disclosed herein. In some embodiments of these methods andkits, the protease is selected from the group consisting of uPA,legumain, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-9, MMP-12,MMP-13, and MMP-14. In some embodiments, the protease is a cathepsin.

Therapeutic Administration and Formulations of Anti-Jagged Antibodies

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

In some embodiments, the anti-Jagged antibodies, the activatableanti-Jagged antibodies and the anti-Jagged antibody compositions used totreat a cancer or fibrotic disorder are administered in conjunction withone or more additional agents, or a combination of additional agents.Suitable additional agents include current pharmaceutical and/orsurgical therapies for an intended application. For example, theanti-Jagged antibodies and/or activatable anti-Jagged antibodies can beused in conjunction with an additional chemotherapeutic oranti-neoplastic agent. For example, the anti-Jagged antibody and/oractivatable anti-Jagged antibody and additional agent are formulatedinto a single therapeutic composition, and the anti-Jagged antibodyand/or activatable anti-Jagged antibody and additional agent areadministered simultaneously. Alternatively, the anti-Jagged antibodyand/or activatable anti-Jagged antibody and additional agent areseparate from each other, e.g., each is formulated into a separatetherapeutic composition, and the anti-Jagged antibody and/or activatableanti-Jagged antibody and the additional agent are administeredsimultaneously, or the anti-Jagged antibody and/or activatableanti-Jagged antibody and the additional agent are administered atdifferent times during a treatment regimen. For example, the anti-Jaggedantibody and/or activatable anti-Jagged antibody is administered priorto the administration of the additional agent, the anti-Jagged antibodyand/or activatable anti-Jagged antibody is administered subsequent tothe administration of the additional agent, or the anti-Jagged antibodyand/or activatable anti-Jagged antibody and the additional agent areadministered in an alternating fashion. As described herein, theanti-Jagged antibody and/or activatable anti-Jagged antibody andadditional agent are administered in single doses or in multiple doses.

In some embodiments, the additional agent is coupled or otherwiseattached to the anti-Jagged antibody and/or activatable anti-Jaggedantibody.

Suitable additional agents are selected according to the purpose of theintended application (i.e., killing, prevention of cell proliferation,hormone therapy or gene therapy). Such agents may include but is notlimited to, for example, pharmaceutical agents, toxins, fragments oftoxins, alkylating agents, enzymes, antibiotics, antimetabolites,antiproliferative agents, hormones, neurotransmitters, DNA, RNA, siRNA,oligonucleotides, antisense RNA, aptamers, diagnostics, radioopaquedyes, radioactive isotopes, fluorogenic compounds, magnetic labels,nanoparticles, marker compounds, lectins, compounds that alter cellmembrane permeability, photochemical compounds, small molecules,liposomes, micelles, gene therapy vectors, viral vectors, and the like.Finally, combinations of agents or combinations of different classes ofagents may be used.

The antibodies and/or activatable antibodies of the invention (alsoreferred to herein as “active compounds”), and derivatives, fragments,analogs and homologs thereof, can be incorporated into pharmaceuticalcompositions suitable for administration. Principles and considerationsinvolved in preparing such compositions, as well as guidance in thechoice of components are provided, for example, in Remington'sPharmaceutical Sciences: The Science And Practice Of Pharmacy 19th ed.(Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995;Drug Absorption Enhancement: Concepts, Possibilities, Limitations, AndTrends, Harwood Academic Publishers, Langhorne, Pa., 1994; and PeptideAnd Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4),1991, M. Dekker, New York.

Such compositions typically comprise the antibody and a pharmaceuticallyacceptable carrier. Where activatable antibody includes a fragment ofthe AB domain, the smallest fragment of the AB that specifically bindsto the binding domain of the target protein can be used. For example,based upon the variable-region sequences of an antibody, peptidemolecules can be designed that retain the ability of the AB to bind thetarget protein sequence. Such peptides can be synthesized chemicallyand/or produced by recombinant DNA technology. (See, e.g., Marasco etal., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).

As used herein, the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Suitable examples ofsuch carriers or diluents include, but are not limited to, water,saline, ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be suitable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas sustained/controlled release formulations, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

For example, the active ingredients can be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

The materials can also be obtained commercially from Alza Corporationand Nova Pharmaceuticals, Inc. Liposomal suspensions (includingliposomes targeted to infected cells with monoclonal antibodies to viralantigens) and can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The formulation can also contain more than one active compound asnecessary for the particular indication being treated, for example,those with complementary activities that do not adversely affect eachother. Alternatively, or in addition, the composition can comprise anagent that enhances its function, such as, for example, a cytotoxicagent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

In one embodiment, the active compounds are administered in combinationtherapy, i.e., combined with other agents, e.g., therapeutic agents,that are useful for treating pathological conditions or disorders, suchas autoimmune disorders and inflammatory diseases. The term “incombination” in this context means that the agents are givensubstantially contemporaneously, either simultaneously or sequentially.If given sequentially, at the onset of administration of the secondcompound, the first of the two compounds is still detectable ateffective concentrations at the site of treatment.

For example, the combination therapy can include one or more antibodiesof the invention coformulated with, and/or coadministered with, one ormore additional therapeutic agents, e.g., one or more cytokine andgrowth factor inhibitors, immunosuppressants, anti-inflammatory agents,metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostaticagents, as described in more detail below. Furthermore, one or moreantibodies described herein may be used in combination with two or moreof the therapeutic agents described herein. Such combination therapiesmay advantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

In other embodiments, one or more antibodies of the invention can becoformulated with, and/or coadministered with, one or moreanti-inflammatory drugs, immunosuppressants, or metabolic or enzymaticinhibitors. Nonlimiting examples of the drugs or inhibitors that can beused in combination with the antibodies described herein, include, butare not limited to, one or more of: nonsteroidal anti-inflammatorydrug(s) (NSAIDs), e.g., ibuprofen, tenidap, naproxen, meloxicam,piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroidssuch. as prednisolone; cytokine suppressive anti-inflammatory drug(s)(CSAIDs); inhibitors of nucleotide biosynthesis, e.g., inhibitors ofpurine biosynthesis, folate antagonists (e.g., methotrexate(N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamicacid); and inhibitors of pyrimidine biosynthesis, e.g., dihydroorotatedehydrogenase (DHODH) inhibitors. Suitable therapeutic agents for use incombination with the antibodies of the invention include NSAIDs, CSAIDs,(DHODH) inhibitors (e.g., leflunomide), and folate antagonists (e.g.,methotrexate).

Examples of additional inhibitors include one or more of:corticosteroids (oral, inhaled and local injection); immunosuppresants,e.g., cyclosporin, tacrolimus (FK-506); and mTOR inhibitors, e.g.,sirolimus (rapamycin—RAPAMUNE™ or rapamycin derivatives, e.g., solublerapamycin derivatives (e.g., ester rapamycin derivatives, e.g.,CCI-779); agents that interfere with signaling by proinflammatorycytokines such as TNFα or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinaseinhibitors); COX2 inhibitors, e.g., celecoxib, rofecoxib, and variantsthereof; phosphodiesterase inhibitors, e.g., R973401 (phosphodiesteraseType IV inhibitor); phospholipase inhibitors, e.g., inhibitors ofcytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketoneanalogs); inhibitors of vascular endothelial cell growth factor orgrowth factor receptor, e.g., VEGF inhibitor and/or VEGF-R inhibitor;and inhibitors of angiogenesis. Suitable therapeutic agents for use incombination with the antibodies of the invention are immunosuppresants,e.g., cyclosporin, tacrolimus (FK-506); mTOR inhibitors, e.g., sirolimus(rapamycin) or rapamycin derivatives, e.g., soluble rapamycinderivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); COX2inhibitors, e.g., celecoxib and variants thereof; and phospholipaseinhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2), e.g.,trifluoromethyl ketone analogs.

Additional examples of therapeutic agents that can be combined with anantibody of the invention include one or more of: 6-mercaptopurines(6-MP); azathioprine sulphasalazine; mesalazine; olsalazine;chloroquine/hydroxychloroquine (PLAQUENIL®); pencillamine;aurothiornalate (intramuscular and oral); azathioprine; coichicine;beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral);xanthines (theophylline, aminophylline); cromoglycate; nedocromil;ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosineagonists; antithrombotic agents; complement inhibitors; and adrenergicagents.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterized herein with respectto Jagged 1 and/or Jagged 2, the design of other therapeutic modalitiesbeyond antibody moieties is facilitated. Such modalities include,without limitation, advanced antibody therapeutics, such as bispecificantibodies, immunotoxins, and radiolabeled therapeutics, generation ofpeptide therapeutics, gene therapies, particularly intrabodies,antisense therapeutics, and small molecules.

For example, in connection with bispecific antibodies, bispecificantibodies can be generated that comprise (i) two antibodies one with aspecificity to Jagged 1 and Jagged 2 and another to a second moleculethat are conjugated together, (ii) a single antibody that has one chainspecific to Jagged 1 and Jagged 2, and a second chain specific to asecond molecule, or (iii) a single chain antibody that has specificityto Jagged 1 and Jagged 2 and a second molecule. Such bispecificantibodies are generated using techniques that are well known forexample, in connection with (i) and (ii) See e.g., Fanger et al. ImmunolMethods 4:72-81 (1994) and Wright et al. Crit, Reviews in Immunol.12125-168 (1992), and in connection with (iii) See e.g., Traunecker etal. Int. J. Cancer (Suppl.) 7:51-52 (1992).

In connection with immunotoxins, antibodies can be modified to act asimmunotoxins utilizing techniques that are well known in the art. Seee.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.5,194,594. In connection with the preparation of radiolabeledantibodies, such modified antibodies can also be readily preparedutilizing techniques that are well known in the art. See e.g., Junghanset al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition,Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat.Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471,and 5,697,902. Each of immunotoxins and radiolabeled molecules would belikely to kill cells expressing Jagged 1, Jagged 2 and/or both Jagged 1and Jagged 2.

In connection with the generation of therapeutic peptides, through theutilization of structural information related to Jagged 1, Jagged 2and/or both Jagged 1 and Jagged 2 and antibodies thereto, such as theantibodies of the invention or screening of peptide libraries,therapeutic peptides can be generated that are directed against Jagged1, Jagged 2 and/or both Jagged 1 and Jagged 2. Design and screening ofpeptide therapeutics is discussed in connection with Houghten et al.Biotechniques 13:412-421 (1992), Houghten PNAS USA 82:5131-5135 (1985),Pinalla et al. Biotechniques 13:901-905 (1992), Blake and Litzi-DavisBioConjugate Chem. 3:510-513 (1992). Immunotoxins and radiolabeledmolecules can also be prepared, and in a similar manner, in connectionwith peptidic moieties as discussed above in connection with antibodies.Assuming that the Jagged 1 molecule, the Jagged 2 molecule and/or boththe Jagged 1 molecule and the Jagged 2 molecule (or a form, such as asplice variant or alternate form) is functionally active in a diseaseprocess, it will also be possible to design gene and antisensetherapeutics thereto through conventional techniques. Such modalitiescan be utilized for modulating the function of Jagged 1, Jagged 2 and/orboth Jagged 1 and Jagged 2. In connection therewith the antibodies ofthe present invention facilitate design and use of functional assaysrelated thereto. A design and strategy for antisense therapeutics isdiscussed in detail in International Patent Application No. WO 94/29444.Design and strategies for gene therapy are well known. However, inparticular, the use of gene therapeutic techniques involving intrabodiescould prove to be particularly advantageous. See e.g., Chen et al. HumanGene Therapy 5:595-601 (1994) and Marasco Gene Therapy 4:11-15 (1997).General design of and considerations related to gene therapeutics isalso discussed in International Patent Application No. WO 97/38137.

Knowledge gleaned from the structure of the Jagged 1 molecule, theJagged 2 molecule and/or both the Jagged 1 molecule and the Jagged 2molecule and its interactions with other molecules in accordance withthe present invention, such as the antibodies of the invention, andothers can be utilized to rationally design additional therapeuticmodalities. In this regard, rational drug design techniques such asX-ray crystallography, computer-aided (or assisted) molecular modeling(CAMM), quantitative or qualitative structure-activity relationship(QSAR), and similar technologies can be utilized to focus drug discoveryefforts. Rational design allows prediction of protein or syntheticstructures that can interact with the molecule or specific forms thereofthat can be used to modify or modulate the activity of Jagged 1, Jagged2 and/or both Jagged 1 and Jagged 2. Such structures can be synthesizedchemically or expressed in biological systems. This approach has beenreviewed in Capsey et al. Genetically Engineered Human Therapeutic Drugs(Stockton Press, NY (1988)). Further, combinatorial libraries can bedesigned and synthesized and used in screening programs, such as highthroughput screening efforts.

Screening Methods

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptides, peptidomimetics, small molecules or otherdrugs) that modulate, block, inhibit, reduce, antagonize, neutralize orotherwise interfere with binding of Jagged 1, Jagged 2 and/or bothJagged 1 and Jagged 2 to their innate receptor, or candidate or testcompounds or agents that modulate, block, inhibit, reduce, antagonize,neutralize or otherwise interfere with the signaling function of Jagged1, Jagged 2 and/or both Jagged 1 and Jagged 2. Also provided are methodsof identifying compounds useful to treat disorders associated withJagged 1, Jagged 2 and/or both Jagged 1 and Jagged 2 signaling. Theinvention also includes compounds identified in the screening assaysdescribed herein.

In one embodiment, the invention provides assays for screening candidateor test compounds that modulate the signaling function of Jagged 1,Jagged 2 and/or both Jagged 1 and Jagged 2. The test compounds of theinvention are obtained using any of the numerous approaches incombinatorial library methods known in the art, including biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds. (See, e.g., Lam, 1997. Anticancer Drug Design 12: 145).

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and in someembodiments less than about 4 kD. Small molecules can be, e.g., nucleicacids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids orother organic or inorganic molecules. Libraries of chemical and/orbiological mixtures, such as fungal, bacterial, or algal extracts, areknown in the art and can be screened with any of the assays of theinvention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (see e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (see Lam, 1991. Nature354: 82-84), on chips (see Fodor, 1993. Nature 364: 555-556), bacteria(see U.S. Pat. No. 5,223,409), spores (see U.S. Pat. No. 5,233,409),plasmids (see Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (see Scott and Smith, 1990. Science 249: 386-390;Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl.Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409).

In one embodiment, a candidate compound is introduced to anantibody-antigen complex and determining whether the candidate compounddisrupts the antibody-antigen complex, wherein a disruption of thiscomplex indicates that the candidate compound modulates the signalingfunction of Jagged 1, Jagged 2 and/or both Jagged 1 and Jagged 2.

In another embodiment, both Jagged 1 and Jagged are provided and exposedto at least one monoclonal antibody. Formation of an antibody-antigencomplex is detected, and one or more candidate compounds are introducedto the complex. If the antibody-antigen complex is disrupted followingintroduction of the one or more candidate compounds, the candidatecompounds is useful to treat disorders associated with Jagged 1, Jagged2 and/or both Jagged 1 and Jagged 2 signaling.

In another embodiment, a soluble protein of the invention is providedand exposed to at least one neutralizing monoclonal antibody. Formationof an antibody-antigen complex is detected, and one or more candidatecompounds are introduced to the complex. If the antibody-antigen complexis disrupted following introduction of the one or more candidatecompounds, the candidate compounds is useful to treat disordersassociated with Jagged 1, Jagged 2 and/or both Jagged 1 and Jagged 2signaling.

Determining the ability of the test compound to interfere with ordisrupt the antibody-antigen complex can be accomplished, for example,by coupling the test compound with a radioisotope or enzymatic labelsuch that binding of the test compound to the antigen orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In one embodiment, the assay comprises contacting an antibody-antigencomplex with a test compound, and determining the ability of the testcompound to interact with the antigen or otherwise disrupt the existingantibody-antigen complex. In this embodiment, determining the ability ofthe test compound to interact with the antigen and/or disrupt theantibody-antigen complex comprises determining the ability of the testcompound to preferentially bind to the antigen or a biologically-activeportion thereof, as compared to the antibody.

In another embodiment, the assay comprises contacting anantibody-antigen complex with a test compound and determining theability of the test compound to modulate the antibody-antigen complex.Determining the ability of the test compound to modulate theantibody-antigen complex can be accomplished, for example, bydetermining the ability of the antigen to bind to or interact with theantibody, in the presence of the test compound.

The screening methods disclosed herein may be performed as a cell-basedassay or as a cell-free assay. The cell-free assays of the invention areamenable to use soluble Jagged 1, Jagged 2 and/or both Jagged 1 andJagged 2, and fragments thereof.

In more than one embodiment, it may be desirable to immobilize eitherthe antibody or the antigen to facilitate separation of complexed fromuncomplexed forms of one or both following introduction of the candidatecompound, as well as to accommodate automation of the assay. Observationof the antibody-antigen complex in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example, GST-antibodyfusion proteins or GST-antigen fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound, and the mixture is incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly.Alternatively, the complexes can be dissociated from the matrix, and thelevel of antibody-antigen complex formation can be determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either theantibody or the antigen (e.g. Jagged 1, Jagged 2 and/or both Jagged 1and Jagged 2) can be immobilized utilizing conjugation of biotin andstreptavidin. Biotinylated antibody or antigen molecules can be preparedfrom biotin-NHS (N-hydroxy-succinimide) using techniques well-knownwithin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, other antibodies reactive withthe antibody or antigen of interest, but that do not interfere with theformation of the antibody-antigen complex of interest, can bederivatized to the wells of the plate, and unbound antibody or antigentrapped in the wells by antibody conjugation. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using such otherantibodies reactive with the antibody or antigen.

The invention further pertains to novel agents identified by any of theaforementioned screening assays and uses thereof for treatments asdescribed herein.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES Example 1 Selection of Human ScFvs of the Embodiments that BindHuman Jagged 1

This Example demonstrates that ScFvs (single-chain variable fragments)of the embodiments that bind Jagged 1 can be selected from a phagedisplay library of ScFvs with diverse CDR sequences, and that suchbinding can be inhibited by Notch 1.

ScFvs were selected from a fully human ScFv library displayed on M13bacteriophage; ScFv phage selection was conducted under contract withCreative Biolabs, Shirley, N.Y.). A fusion protein comprised of theextracellular domain (ECD) of human Jagged 1 fused to the Fc portion ofhuman IgG1 (R&D Systems, Minneapolis, Minn., Cat#1277-JG-050) was usedas the antigen in three rounds of selection for ScFvs displayed on M13bacteriophage that bind human Jagged 1. All selections were done in thepresence of CA₂ ⁺⁺, required for the native conformation of Jagged 1,and human IgG1 to prevent human Fc binding. In the first round, boundphage were released by trypsin digestion, and in subsequent rounds,phage were eluted by human Notch 1-Fc fusion protein (R&D Systems; Cat#3637-TK-050) competition. Five (5) unique ScFvs that bind human Jagged1were isolated. Table 1 lists the 5 ScFvs and SEQ ID NOs of theirrespective nucleic acid sequences and amino acid sequences.

TABLE 1 SEQ ID NOs of selected ScFvs ScFv Nucleic acid sequence Aminoacid sequence Jagged 2 SEQ ID NO: 1 SEQ ID NO: 2 Jagged 7 SEQ ID NO: 3SEQ ID NO: 4 Jagged 13 SEQ ID NO: 5 SEQ ID NO: 6 Jagged 42 SEQ ID NO: 7SEQ ID NO: 8 Jagged 32 SEQ ID NO: 9 SEQ ID NO: 10

The nucleic acid and amino acid sequences of each of the anti-JaggedScFvs are shown below:

SEQ ID NO: 1gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggagggctggagtgggtctcagcgattgcggagctgggtgcgcttacatagtacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagctagagccgaggacacggccgtatattactgtgcgagagctcatactagttttgactactggggccagggaaccctggtcaccgtctcgagcggtggaggcggttcaggcggaggtggcagcggcgggggggtcgacggacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaccgggaaagcccctaagctcctgatctataaggcatccactttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaaatttgcaacttactactgtcaacaggctatggatcagcctcctacgttcggccaagggaccaaggtggaaatcaaacgg-3′SEQ ID NO: 2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIAELGALTQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAHTSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYKASTLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAMDQPPTFGQGTKVE IKR SEQ IDNO: 3gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggagggctggagtgggtctcaacgattgctgcttagggtaagcatacagattacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagctagagccgaggacacggccgtatattactgtgcgaaatcgatgcgtggttttgacaactggggccagggaaccctggtcaccgtctcgagcggtggaggcggttcaggcggaggtggcagcggcgggggggtcgacggacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaccgggaaagcccctaagctcctgatctatcgggcatcctctttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaaatttgcaacttactactgtcaacaggatgcgactggtcctgcgacgttcggccaagggaccaaggtggaaatcaaacgg-3′SEQ ID NO: 4 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIAA*GKHTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSMRGFDNWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDATGPATFGQGTKV EIKR SEQID NO: 5gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggagggctggagtgggtctcatcgattgagacttagggtccgactacactgtacgcagactccgtgaagggcaggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagctagagccgaggacacggccgtatattactgtgcgaaaacgtctagtgcgtttgactactggggccagggaaccctggtcaccgtctcgagcggtggaggcggttcaggcggaggtggcagcggcgggggggtcgacggacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaccgggaaagcccctaagctcctgatctatcatgcatcctcgttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcggcagtctgcaacctgaaatttgcaacttactactgtcaacagaatgttgctactcctctgacgttcggccaagggaccaaggtggaaatcaaacgg-3′SEQ ID NO: 6 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIETQGPTTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSSAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYASSLQSGVPSRFSGSGSGTDFTLTIGSLQPEDFATYYCQQNVATPLTFGQGTKVEI KR SEQ IDNO: 7gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggagggctggagtgggtctcaacgattgagccgtagggttcggctacagagtacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagctagagccgaggacacggccgtatattactgtgcgaaaacgcagacgggttttgactactggggccagggaaccctggtcaccgtctcgagcggtggaggcggttcaggcggaggtggcagcggcgggggggtcgacggacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaccgggaaagcccctaagctcctgatctataaggcatccactttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaaatttgcaacttactactgtcaacaggatgttgagcctcctgctacgttcggccaagggaccaaggtggaaatcaaacgg-3′SEQ ID NO: 8 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIEP*GSATEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTQTGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDVEPPATFGQGTKVE IKR SEQ IDNO: 9Gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggagggctggagtgggtctcaagtattgagcagatgggttggtagacatattacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagctagagccgaggacacggccgtatattactgtgcgaaatcggctgctgcttttgactactggggccagggaaccctggtcaccgtctcgagcggtggaggcggttcaggcggaggtggcagcggcgggggggtcgacggacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaccgggaaagcccctaagctcctgatctatgcggcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaaatttgcaacttactactgtcaacagacggttgtggcgcctttgacgttcggccaagggaccaaggtggaaatcaaacgg-3′SEQ ID NO: 10 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPLTFGQGTKV EIKR

ELISA-based binding of Jagged 13 ScFv-phage and Jagged 32 ScFv-phage tohuman Jagged 1 was shown to be inhibited by human Notch 1. Briefly,human Jagged 1-Fc (R&D Systems; ibid.) was adsorbed to the wells of a96-well ELISA plate. Phage were applied to the plate in the presence orabsence of human notch 1-Fc and allowed to bind. Bound phage werevisualized with an anti-M13-HRP conjugate (GE Healthcare, Piscataway,N.Y.) and developed with the chromogenic substrate tetramethyl benzidine(TMB) (Thermo Scientific, Rockford, Ill.).

Example 2 Production and Testing of Fully Human Jagged IgG Antibodies ofthe Embodiments

This Example demonstrates that Jagged ScFv-phage that bind human Jagged1 can be converted into fully human IgG antibodies that bind both humanJagged 1 and human Jagged 2, as well as mouse Jagged 1. Human Notch 1can inhibit such binding.

Production of fully human IgGs comprising the variable domains of Jagged13 and Jagged 32 was accomplished using techniques similar to thosedescribed in PCT Publication No. WO 2010/081173, ibid. DNA encoding theJagged 13 and 32 variable domains of Jagged 13 ScFv-phage and Jagged 32ScFv-phage were cloned into expression vectors for the expression offully human IgGs. Light chain (Lc) variable domains were amplified fromthe ScFv templates using primer CX1197(cacttgtcacgaattcggacatccagatgacccagtc) (SEQ ID NO: 83) and primerCX1198 (gtgcagccaccgtacgtttgatttccaccttggtccc) (SEQ ID NO: 84). Vector(LcpOP (modified from pcDNA3, Invitrogen, Carlsbad, Calif.)) andamplified DNA were cut with BsiWI and EcoRI overnight, combined byligation and transformed into E. coli MC1061 cells. Heavy chain (Hc)variable domains were amplified from ScFv templates using primer CX1199(ttgcacttgtcacgaattcggaggtgcagctgttggagtc) (SEQ ID NO: 85) and primerCX1202 (ggcccttggtgctagcgctcgagacggtgaccagggttc) (SEQ ID NO: 86). DNAencoding the interleukin 2 (IL2) signal sequence was amplified fromHcpPOP (modified from pcDNA3, Invitrogen) using primer CX1184(gaaccgtcagatcactagaagc) (SEQ ID NO: 81) and primer CX1185(cgaattcgtgacaagtgcaagacttagtg) (SEQ ID NO: 82), and annealed with theHc variable domains. Vector (HcpOP (modified from pcDNA3, Invitrogen))and the IL2-Hc-variable domains were cleaved with HindIII and NheIovernight, combined by ligation and transformed into E. coli MC1061cells. Fully human IgGs (i.e., Jagged 13 IgG and Jagged 32 IgG, alsoreferred to herein as anti-Jagged 13 (or anti-Jag 13) and anti-Jagged 32(or anti-Jag 32), respectively) were expressed from transientlytransfected HEK-293 cells and purified from the culture supernatant byProtein A chromatography.

As shown in FIG. 1, ELISA-binding experiments revealed that anti-Jagged13 IgG and anti-Jagged 32 IgG bound human and mouse Jagged 1 and humanJagged 2, with affinities above 30 nM: Human Jagged 1-Fc (R&D Systems;ibid.), human Jagged 2-Fc (R&D Systems; Cat #1726-JG-050), or rat Jagged1-Fc (R&D Systems; Cat #599-JG-100) was adsorbed to the wells of a96-well ELISA plate. Purified anti-Jagged 13 and anti-Jagged 32antibodies were applied to the plate and allowed to bind. Bound antibodywas visualized with an anti-human IgG-HRP conjugate, Fab specific,(Sigma, St Louis, Mo.; Cat # A0293-1ML) and developed with thechromogenic substrate TMB.

As shown in FIG. 2, ELISA-binding experiments demonstrated that Jagged 1binding by anti-Jagged 13 and anti-Jagged 32 binding was inhibited byhuman Notch 1: For the competition experiments, Notch 1-Fc (R&D Systems;ibid.) was adsorbed to the wells of a 96-well ELISA plate. Biotinylatedhuman Jagged 1-Fc (R&D Systems; ibid.) was applied to the plate inincreasing concentrations of anti-Jagged 13 and anti-Jagged 32antibodies, and allowed to bind. Bound Jagged 1 was visualized withStreptavidin-HRP (Thermo Scientific) and developed with the chromogenicsubstrate TMB. Anti-Jagged MAB-12771 (R&D Systems; Cat# MAB12771) wasused as a positive control for inhibition and bevacizumab as a negativecontrol. The terms “old” and “new” refer to different lots of antibodyproduction. Antibody 32/13 has an anti-Jagged 32 light chain and ananti-13 heavy chain. Antibody 32/32 has anti-Jagged 32 light and heavychains.

Example 3 Affinity Maturation of Anti-Jagged Antibodies of theEmbodiments

This Example demonstrates the isolation of antibodies of the embodimentswith improved binding kinetics and Jagged binding specificities.

Anti-Jagged antibodies were isolated from libraries with CDRs modifiedfrom anti-Jagged 32. Such libraries were designed as shown in Table 2.Six libraries of antibodies, based on the sequence of anti-Jagged 32,were constructed using Dut/Ung mutagenesis (see, e.g., Kunkel T A, 1985,Proc Natl Acad Sci 82, 488-492). Residues were either varied by softrandomization at each indicated nucleotide, by retaining 70% of theoriginal nucleotide and 10% of each of the other three possiblenucleotides (superscript 1 in Table 2), or by total randomization(superscript 2 in Table 2). In addition, within libraries 3 and 6,additional residues were added to CD3 of the heavy chain. Libraries weretransfected into E. coli strain TG1 and phage were prepared followingsuper-infection with M13KO7 (Invitrogen).

Three rounds of selection were performed for each library withincreasing stringency. For round three, human Jagged 1 (R&D Systems;ibid.) was adsorbed to immunotubes (Nunc, Denmark) at 5 micrograms perml (μg/mL). Phage were blocked with 100 μg/mL pooled human IgG (huIgG,or hIgG) and 2% non-fat dried milk (NFDM) in Tris-buffered saline (TBS;40 mM Tris, 129 mM NaCl, pH 7.4), and then added to the coated tubes forbinding. Following binding, the tubes were washed extensively includingfour 37° C. washes for 30 minutes each. Following the washes theremaining bound phage were eluted with 100 mM triethanolamine (TEA)(Sigma, St. Louis, Mo.) and expanded through E. coli TG1. Libraries 1, 2and 5 were combined to form library 125, and libraries 3, 4 and 6 werecombined to form library 346; each library was subjected to anadditional round of selection, also referred to herein as round fourselection of library 125 and round four selection of library 346,respectively, as described for round three.

TABLE 2 CDR sequences for affinity maturation libraries Lib # CDR1 CDR2CDR3 1 Light RASQSISSLYN AASLQS (SEQ ID NO: 198) QQT¹V¹V¹A¹PL¹T¹ chain(SEQ ID NO: (SEQ ID NO: 199) 197) Heavy SAYMS (SEQ SIEQMGGWQTYYADSSVKGSAAAFDY (SEQ ID chain ID NO: 200) (SEQ ID NO: 201) NO: 202) 2 LightRASQSISSLYN AASLQS (SEQ ID NO: 198) QQTVVAPLT (SEQ ID chain (SEQ ID NO:NO: 203) 197) Heavy SAYMS (SEQ S¹IE¹Q¹M¹GGW¹Q¹TYYADSSVKG SAAAFDY (SEQ IDchain ID NO: 200) (SEQ ID NO: 204) NO: 202) 3 Light RASQSISSLYN AASLQS(SEQ ID NO: 198) QQTVVAPLT (SEQ ID chain (SEQ ID NO: NO: 203) 197) HeavySAYMS (SEQ SIEQMGGWQTYYADSSVKG SA²A²A²FDYXXXX chain ID NO: 200) (SEQ IDNO: 201) (SEQ ID NO: 205) 4 Light RASQSISSLYN AASLQS (SEQ ID NO: 198)QQTVVAPLT (SEQ ID chain (SEQ ID NO: NO: 203) 197) Heavy SAYMS (SEQS¹IE¹Q¹M¹GGW¹Q¹TYYADSSVKG SA²A²A²FDY (SEQ ID chain ID NO: 200) (SEQ IDNO: 204) NO: 206) 5 Light RASQSISSLYN AASLQS (SEQ ID NO: 198)QQT¹V¹V¹A¹PL¹T¹ chain (SEQ ID NO: SEQ ID NO: 199) 197) Heavy SAYMS (SEQSIEQMGGWQTYYADSSVKG SA²A²A²FDY (SEQ ID chain ID NO: 200) (SEQ ID NO:201) NO: 206) 6 Light RASQSISSLYN AASLQS (SEQ ID NO: 198) QQTVVAPLT (SEQID chain (SEQ ID NO: NO: 203) 197) Heavy SAYMS (SEQ SIEQMGGWQTYYADSSVKGSA²A²A²FDYXXXXxxx chain ID NO: 200) (SEQ ID NO: 201) (SEQ ID NO: 207)Superscript 1 denotes residues that were varied by soft randomization ateach indicated nucleotide, by retaining 70% of the original nucleotideand 10% of each of the other three possible nucleotides, whilesuperscript 2 denotes residues that were varied by total randomization.

Example 4 Binding Characteristics of Affinity Matured Anti-JaggedAntibodies

This Example demonstrates the binding characteristics ofaffinity-matured anti-Jagged antibodies of the embodiments isolated fromaffinity maturation processes.

Forty-eight (48) clones from each round four selection of library 125and round four selection of library 346 were grown and infected withM13KO7 to generate phage. Each phage was analyzed for its ability tobind human Jagged 1-Fc (R&D Systems; ibid.), rat Jagged 1-Fc (R&DSystems; ibid.), and human Jagged 2-Fc (R&D Systems; ibid.) by phageELISA. Jagged ligands were adsorbed to the wells of a 96-well ELISAplate, each ligand on a separate plate. Phage were applied tocorrelative wells on each plate and allowed to bind. Bound phage werevisualized with an anti-M13-HRP conjugate and developed with thechromogenic substrate TMB. Individual isolates displayed divergentbinding specificities; these specificities are shown in Table 3. Alsoshown are the SEQ ID NOs for the each of these isolates.

TABLE 3 Unique isolates have distinct binding specificities SEQ ID NO ofSEQ ID NO of heavy light chain amino chain amino acid hJag1 hJag2 rJag1acid sequence sequence 346.4 ++ ++ SEQ ID NO: 11 SEQ ID NO: 12 346.5 ++++ SEQ ID NO: 13 SEQ ID NO: 14 346.7 +++ +++ +++ SEQ ID NO: 15 SEQ IDNO: 16 346.8 + ++ SEQ ID NO: 17 SEQ ID NO: 18 346.13 ++ SEQ ID NO: 19SEQ ID NO: 20 346.16 +++ + +++ SEQ ID NO: 21 SEQ ID NO: 22 346.19 + ++SEQ ID NO: 23 SEQ ID NO: 24 346.21 + +/− SEQ ID NO: 25 SEQ ID NO: 26346.24 + + +/− SEQ ID NO: 27 SEQ ID NO: 28 346.26 ++ + SEQ ID NO: 29 SEQID NO: 30 346.27 ++ ++ SEQ ID NO: 31 SEQ ID NO: 32 346.28 +++ +++ +++SEQ ID NO: 33 SEQ ID NO: 34 346.30 ++ SEQ ID NO: 35 SEQ ID NO: 36 346.31++ + SEQ ID NO: 37 SEQ ID NO: 38 346.32 + SEQ ID NO: 39 SEQ ID NO: 40346.37 + SEQ ID NO: 41 SEQ ID NO: 42 346.39 + +++ SEQ ID NO: 43 SEQ IDNO: 44 346.40 +++ ++ +++ SEQ ID NO: 45 SEQ ID NO: 46 346.47 +++ SEQ IDNO: 47 SEQ ID NO: 48

The amino acid sequences of each of the clones in Table 3 are shownbelow:

Lc4 SEQ ID NO: 11 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc4 SEQ IDNO: 12 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DIGGRSAFDYWGQGTLVTVSSLc5 SEQ ID NO: 13 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc5 SEQ IDNO: 14 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SPPYHGQFDYWGQGTLVTVSSLc7 SEQ ID NO: 15 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc7 SEQ IDNO: 16 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SPPFFGQFDYWGQGTLVTVSSLc8 SEQ ID NO: 17 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc8 SEQ IDNO: 18 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK HIGRTNPFDYWGQGTLVTVSSLc13 SEQ ID NO: 19 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc13 SEQ IDNO: 20 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SAAAFDYWGQGTLVTVSSLc16 SEQ ID NO: 21 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc16 SEQ IDNO: 22 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SPPYYGQFDYWGQGTLVTVSSLc19 SEQ ID NO: 23 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc19 SEQ IDNO: 24 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SPPFFGQFDYWGQGTLVTVSSLc21 SEQ ID NO: 25 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc21 SEQ IDNO: 26 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DIGGRSAFDYWGQGTLVTVSSLc24 SEQ ID NO: 27 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc24 SEQ IDNO: 28 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SAAAFDYWGQGTLVTVSSLc26 SEQ ID NO: 29 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc26 SEQ IDNO: 30 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DIGGRSAFDYWGQGTLVTVSSLc27 SEQ ID NO: 31 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc27 SEQ IDNO: 32 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SPPFYGQFDYWGQGTLVTVSSLc28 SEQ ID NO: 33 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc28 SEQ IDNO: 34 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SPPFFGQFDYWGQGTLVTVSSLc30 SEQ ID NO: 35 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc30 SEQ IDNO: 36 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYAKS AAAFDYWGQGTLVTVSS Lc31SEQ ID NO: 37 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc31 SEQ IDNO: 38 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DIGGRSAFDYWGQGTLVTVSSLc32 SEQ ID NO: 39 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc32 SEQ IDNO: 40 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SAAAFDYWGQGTLVTVSSLc37 SEQ ID NO: 41 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc37 SEQ IDNO: 42 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SPPHNGQFDYWGQGTLVTVSSLc39 SEQ ID NO: 43 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc39 SEQ IDNO: 44 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SAAAFDYWGQGTLVTVSSLc40 SEQ ID NO: 45 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc40 SEQ IDNO: 46 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SPPFFGQFDYWGQGTLVTVSSLc47 SEQ ID NO: 47 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTF GQGTKVEIKR Hc47 SEQ IDNO: 48 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SAAAFDYWGQGTLVTVSS

Example 5 Isolation and Testing of Affinity Matured Anti-Jagged 1 andAnti-Jagged 2 Antibodies of the Embodiments

This Example describes the use of CDR shuffling to isolate antibodies ofthe embodiments that exhibit enhanced binding affinities for Jagged 1and/or Jagged 2.

A seventh library was constructed by combining the light chains from theround four selection of library 125 and the heavy chains from the roundfour selection of library 346. This H/L library was selected through twoadditional rounds. In round one, the library was divided in two parts:one part was selected for binding to human Jagged 1-Fc (R&D Systems;ibid.) and the second part was selected for binding to human Jagged 2-Fc(R&D Systems, ibid.). In round two, the human Jagged 1-selected phagefrom round 1 were selected for binding to human Jagged 1-Fc (R&DSystems; ibid.) or human Jagged 2-Fc (R&D Systems, ibid.) in separatebinding reactions, yielding two pools, designated Jagged 1/1 and Jagged1/2, respectively. Similarly, the human Jagged 2-selected phage fromround one were selected for binding to human Jagged 1-Fc (R&D Systems;ibid.) or human Jagged 2 (R&D Systems; ibid.) in separate bindingreactions, yielding two pools, designated Jagged 2/1 and Jagged 2/2,respectively.

Ninety-five (95) individual isolates were chosen from each of the fourpools. Phage were derived from each isolate and assayed for binding tohuman Jagged 1-Fc (R&D Systems; ibid.) or human Jagged 2-Fc (R&DSystems; ibid). The Jagged ligands were adsorbed to the wells of a96-well ELISA plate, each ligand on a separate plate. Phage were appliedto correlative wells on each plate and allowed to bind. Bound phage werevisualized with an anti-M13-HRP conjugate and developed with thechromogenic substrate TMB. Based on the results of the ELISA and DNAsequence, 6 unique clones were chosen for further study. Table 4 liststhe antibodies encoded by the 6 clones and SEQ ID NOs for the nucleicacid sequences and amino acid sequences of their respective light chainsand heavy chains.

TABLE 4 SEQ ID NOs of the six clones encoding affinity maturedantibodies Light chain Heavy chain Amino Nucleic Nucleic acid acid acidAmino acid Antibody sequence sequence sequence sequence 4B2 SEQ ID NO:49 SEQ ID SEQ ID SEQ ID NO: 52 NO: 50 NO: 51 4D11 SEQ ID NO: 53 SEQ IDSEQ ID SEQ ID NO: 56 NO: 54 NO: 55 4E7 SEQ ID NO: 57 SEQ ID SEQ ID SEQID NO: 60 NO: 58 NO: 59 4E11 SEQ ID NO: 61 SEQ ID SEQ ID SEQ ID NO: 64NO: 62 NO: 63 6B7 SEQ ID NO: 65 SEQ ID SEQ ID SEQ ID NO: 68 NO: 66 NO:67 6F8 SEQ ID NO: 69 SEQ ID SEQ ID SEQ ID NO: 72 NO: 70 NO: 71

The amino acid sequences of each of the final clones in Table 4 afterchain shuffle are shown below:

4B2 Light Chain SEQ ID NO: 49GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGCTAGACGCTCCTCCGCAATTCGGCCAAGGGACCAAGGTGGAAATCAAACGT SEQ ID NO: 50 D I Q M T Q S P S S L S A SV G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y AA S S L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D F A T YY C Q Q T L D A P P Q F G Q G T K V E I K R Heavy Chain SEQ ID NO: 51GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGAGCAGATGGGTTGGCAGACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACATCGGCGGCAGGTCGGCCTTTGACTACTGGGGCCAGGGAACCCTGG TCACCGTCTCCTCA SEQ IDNO: 52 E V Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S Y AM S W V R Q A P G K G L E W V S S I E Q M G W Q T Y Y A D S V K G R F TI S R D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K D I G G R S AF D Y W G Q G T L V T V S S 4D11 Light Chain SEQ ID NO: 53GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGT SEQ ID NO: 54 D I Q M T Q S P S S L S A SV G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y AA S S L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D F A T YY C Q Q T V V A P P L F G Q G T K V E I K R Heavy Chain SEQ ID NO: 55GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAAGTATTGACCCGGAAGGTCGGCAGACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACATCGGCGGCAGGTCGGCCTTTGACTACTGGGGCCAGGGAACCCTGG TCACCGTCTCCTCA SEQ IDNO: 56 E V Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S Y AM S W V R Q A P G K G L E W V S S I D P E G R Q T Y Y A D S V K G R F TI S R D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K D I G G R S AF D Y W G Q G T L V T V S S 4E7 Light Chain SEQ ID NO: 57GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCGCTGGTGGCGCCTCTTACCTTCGGCCAAGGGACCAAGGTGGAAATCAAACGT SEQ ID NO: 58 D I Q M T Q S P S S L S A SV G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y AA S S L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D F A T YY C Q Q S L V A P L T F G Q G T K V E I K R Heavy Chain SEQ ID NO: 59GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAAGTATTGAAGAGATGGGTTGGCAGACAAAGTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCGGCTGCTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA SEQ ID NO: 60 EV Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S Y A M S W VR Q A P G K G L E W V S S I E E M G W Q T K Y A D S V K G R F T I S R DN S K N T L Y L Q M N S L R A E D T A V Y Y C A K S A A A F D Y W G Q GT L V T V S S 4E11 Light Chain SEQ ID NO: 61GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGCGTTAGATGCCCCTCTGATGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGT SEQ ID NO: 62 D I Q M T Q S P S S L S A SV G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y AA S S L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D F A T YY C Q Q A L D A P L M F G Q G T K V E I K R Heavy Chain SEQ ID NO: 63GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAAGTATTGAGCCTATGGGTTGACTAACAGAATACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACATCGGCGGCAGGTCGGCCTTTGACTACTGGGGCCAGGGAACCCTGG TCACCGTCTCCTCA SEQ IDNO: 64 E V Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S Y AM S W V R Q A P G K G L E W V S S I E P M G Q L T E Y A D S V K G R F TI S R D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K D I G G R S AF D Y W G Q G T L V T V S S 6B7 Light Chain SEQ ID NO: 65GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGCGCTTGTCGCCCCTCTGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGT SEQ ID NO: 66 D I Q M T Q S P S S L S A SV G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y AA S S L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D F A T YY C Q Q A L V A P L T F G Q G T K V E I K R Heavy Chain SEQ ID NO: 67GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAAGTATTGATGAGATGGGTTGGCAGACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCGGCTGCTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA SEQ ID NO: 68 EV Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S Y A M S W VR Q A P G K G L E W V S S I D E M G W Q T Y Y A D S V K G R F T I S R DN S K N T L Y L Q M N S L R A E D T A V Y Y C A K S A A A F D Y W G Q GT L V T V S S 6F8 Light Chain SEQ ID NO: 69GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGCGCTTGTCGCCCCTCTGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTAC SEQ ID NO: 70 D I Q M T Q S P S S L S AS V G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I YA A S S L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D F A TY Y C Q Q A L V A P L T F G Q G T K V E I K R Heavy Chain SEQ ID NO: 71GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAAGTATTGATGAGATGGGTTGGCAGACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCGGCTGCTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA SEQ ID NO: 72 EV Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S Y A M S W VR Q A P G K G L E W V S S I D E M G W Q T Y Y A D S V K G R F T I S R DN S K N T L Y L Q M N S L R A E D T A V Y Y C A K S A A A F D Y W G Q GT L V T V S S

The libraries were configured with a His tag carboxy to the Fab, and anamber stop codon carboxy to the His tag, such that, when the phagemidsencoding the six affinity matured antibodies were in a non-ambersuppressor strain, the phagemids directed the expression of a C-terminalHis tagged Fab that could be purified from the periplasmic space of E.coli. To measure the affinities of the six matured isolates, as well asthe affinities of anti-Jagged 13 and anti-Jagged 32 Fabs, Fabs wereexpressed and purified from E. coli DH12b.

Off rates for individual Fabs were measured using an Octet (ForteBio,Menlo Park, Calif.). Anti-human Fc Octet tips (ForteBio, Cat #18-5060)were blocked with biocytin and 100 μg/mL BSA and then loaded with 25micromolar (25 μM) Jagged ligand, namely human Jagged 1-Fc (R&D Systems;ibid.), human Jagged2-Fc (R&D Systems; ibid.) or murine Jagged 2-Fc (R&DSystems, ibid.). Following a wash, the loaded tips were exposed to 25 μMFab until binding had reached equilibrium. The tips were then removed toa fresh solution with no Fab, and the rate of Fab dissociation wasmeasured. The dissociation constants listed in Table 5 show that theoff-rates of the affinity matured antibodies have been decreased 10 to100 fold compared to ScFv antibodies Jagged 13 and Jagged 32.

TABLE 5 Dissociation constants for anti-Jagged Fabs k_(diss) (s⁻¹)Antibody h Jagged 1 h Jagged 2 m Jagged 2 4B2   5 × 10⁻⁴ 6.5 × 10⁻³ 1.1× 10⁻³ 4D11 1.2 × 10⁻⁴ 4.5 × 10⁻⁴ 4.2 × 10⁻⁴ 4E7 6.9 × 10⁻⁴ 1.9 × 10⁻³5.8 × 10⁻³ 4E11 1.4 × 10⁻³ 5.7 × 10⁻⁴ 2.5 × 10⁻⁵ 6B7 3.5 × 10⁻⁴   2 ×10⁻³ 2.4 × 10⁻³ 6F8 1.2 × 10⁻³ 4.9 × 10⁻³ 1.6 × 10⁻³ Jag13 2.2 × 10⁻²  1 × 10⁻³ 2.8 × 10⁻³ Jag32 2.7 × 10⁻² 2.3 × 10⁻³ 2.5 × 10⁻³

Example 6 Production of Anti-Jagged Antibodies of the Embodiments

This Example demonstrates the expression and purification of anti-Jaggedantibodies of the embodiments.

Vectors were made in the following manner: The IL2 signal sequencecoding region was moved from pINFUSE-hIgG1-Fc2 (InvivoGen, San Diego,Calif.) as a KasI/NcoI fragment to pFUSE2-CLIg-hk (InvivoGen) digestedwith KasI/NcoI, resulting in plasmid pFIL2-CL-hk. The IL2 signalsequence coding region was also moved from pINFUSE-hIgG1-Fc2 as aKasI/EcoRI fragment to pFUSE-CHIg-hG1 (InvivoGen) digested withKasI/EcoRI (large and medium fragments) in a three-way ligation,resulting in plasmid pFIL-CHIg-hG1.

The light chain coding region from the pFIL2-CL-hk vector was amplifiedusing primers CX1170 and CX1168 and cloned into the pOP Neo vector usingthe NheI and NotI sites using the infusion cloning system (HD EcoDry,Clontech, Mountain View, Calif.), mutating the NotI site in the process.The 4D11 variable light chain coding region was PCR amplified from theisolated 4D11 coding sequence using primers CX1197 and CX1198 and clonedinto the EcoRI and BsiWI restriction sites. The primers are providedTable 6. The 4D11 light chain nucleic acid sequence is represented bySEQ ID NO:73, and the amino acid sequence is represented by SEQ IDNO:74.

4D11 Light Chain sequence: SEQ ID NO: 73gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgcggcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagacggttgtggcgcctccgttattcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggag agtgt SEQ ID NO: 74DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

The heavy chain coding region from the pFIL-CHIg-hG1 vector was clonedinto the pOP Hygr vector (modification of pcDNA3, Invitrogen) asfollows: Two over lapping fragments were amplified, the first was the 5′non-coding region from the pOP Hygr vector using primers CX1184 andCX1185 and the second was the coding region from pFIL-CHIg-hG1 usingprimers CX1172 and CX1169. These two PCR products were then combined fora final amplification using primers CX1184 and CX1169 and cloned intothe pOP Hygr vector using the HindIII and NotI restriction sites. The4D11 variable heavy chain coding region was cloned in a similar way,using the same first DNA fragment and the second fragment beingamplified from the isolated 4D11 coding sequence using primers CX1199and CX1202. The two fragments were amplified using primers CX1184 andCX1202 and cloned into the HindIII and NheI restriction sites. Theprimers are provided in Table 6. The 4D11 light chain nucleic acidsequence is represented by SEQ ID NO:75, and the amino acid sequence isrepresented by SEQ ID NO:76.

4D11 Heavy Chain sequence: SEQ ID NO: 75gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtgtcaagtattgacccggaaggtcggcagacatattacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaaagacatcggcggcaggtcggcctttgactactggggccagggaaccctggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa SEQ ID NO: 76EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

TABLE 6 Primer sequences Primer Nucleic acid sequence SEQ ID NO CX1168tagactcgagcggccgcctaacac SEQ ID NO: 77 tctcccctgttgaagc CX1169tagactcgagcggccgctcatttaccc SEQ ID NO: 78 ggagacagggag CX1170ctcactataggctagcgccaccatgt SEQ ID NO: 79 acaggatgcaactc CX1172ctcactataggctagagccaccatgtac SEQ ID NO: 80 aggatgcaactc CX1184gaaccgtcagatcactagaagc SEQ ID NO: 81 CX1185cgaattcgtgacaagtgcaagacttagtg SEQ ID NO: 82 CX1197cacttgtcacgaattcggacatccagatg SEQ ID NO: 83 acccagtc CX1198gtgcagccaccgtacgtttgatttccacc SEQ ID NO: 84 ttggtccc CX1199ttgcacttgtcacgaattcggaggtgcagc SEQ ID NO: 85 tgttggagtc CX1202ggcccttggtgctagcgctcgagacggtgac SEQ ID NO: 86 cagggttc

Fully human IgGs were expressed from transiently transfected HEK-293cells and purified from the culture supernatant by Protein Achromatography.

Example 7 An Anti-Jagged Antibody of the Embodiments Reduces BxPC-3Tumors in Mice

In this Example, anti-Jagged 4D11 was analyzed for the ability to reducethe growth of BxPC-3 xenograft tumors.

The human pancreatic cancer cell line BxPC-3 was obtained from Cell Bankof Shanghai Institute for Biological Sciences, Chinese Academy ofSciences. The BxPC-3 xenografts were developed by injecting BxPC-3 cellssubcutaneously into the right flank of Balb/c nude mice. Upon reaching500-700 mm3, the tumor was harvested for in vitro cell culture andserial passage. The in vivo adapted BxPC-3 cells (xenograft derivedcells) were grown in RPMI-1640 supplemented with 10% fetal bovine serumat 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells wereroutinely subcultured twice weekly. Cells were harvested during thelogarithmic growth period, resuspended in physical PBS with proper cellconcentration, and kept on ice for tumor induction.

Each mouse was inoculated subcutaneously at the right flank with 5×10⁶of BxPC-3 cells in 0.1 ml of PBS for tumor development. The treatmentswere started when the mean tumor size reached approximately 150 mm³.Tumor sizes were measured twice weekly in two dimensions using acaliper, and the volume was expressed in mm³ using the formula: V=0.5a×b² where a and b are the long and short diameters of the tumor,respectively.

The mice were grouped and dosed as set forth in Table 7.

TABLE 7 Groups and doses for BxPC-3 xenograft study Dose Dosing Group nTreatment (mg/kg) Route Schedule 1 8 IVIg 20 mg/kg i.p q3dx 4 (CTX000) 26 CTX-014 20 mg/kg i.p. q3dx 4 (4D11)

FIG. 3, which plots tumor volume versus number of days post initialdose, demonstrates that anti-Jagged AD11 antibody inhibits the growth ofBxPC-3 xenograft tumors. FIG. 4 indicates weight loss by animals inGroups 1 and 2. The serum concentration of mouse thymic stromallymphopoietin (TSLP) was measured using the Quantikine mouse TSLPimmunoassay (R&D Systems) following the manufacturer's protocol. Theserum levels of mouse TSLP was quantified for individual mice from eachgroup and averaged to generate FIG. 5.

Anti-Jagged 4D11 was also tested for the ability to reduce the growth ofBxPC-3 xenograft tumors in a dose dependent matter, using a methodsimilar to that described above, using the groups and doses set forth inTable 8.

TABLE 8 Groups and doses for dose dependent BxPC-3 xenograft study DoseDosing Group n Treatment (mg/kg) Route Schedule 1 8 PBS 20 mg/kg i.pq3dx 4 2 8 4D11 20 mg/kg i.p. q7dx 4 3 8 4D11 6.7 mg/kg  i.p. q7dx 4 4 84D11  2 mg/kg i.p. q7dx 4 5 8 Gemcitabine 100 mg/kg  i.p. qdx 4

FIG. 6, which plots tumor volume versus number of days post initialdose, demonstrates that anti-Jagged AD11 antibody inhibits the growth ofBxPC-3 xenograft tumors. FIG. 7 indicates weight loss by the animals.

In a second study, anti-Jagged 4D11 was also tested for the ability toreduce the growth of BxPC-3 xenograft tumors in combination with asecond anti-cancer agent. In this study, anti-Jagged 4D11 wasadministered alone or in combination with gemcitabine, the currentstandard-of-care chemotherapy in pancreatic cancer, using a methodsimilar to that described above, and using the doses set forth in FIG.23. In these studies, antibody toxicity was apparent from weight lossand mortality. These studies demonstrate that the combination ofanti-Jagged 4D11 and gemcitabine inhibits the growth of BxPC-3 xenografttumors. As seen in FIG. 23, the combination of anti-Jagged antibody andgemcitabine produced an additive effect in the BXPC3 pancreaticxenograft model.

Example 8 An Anti-Jagged Antibody of the Embodiments Inhibits the Growthof RPMI 8226 in Human Bone Marrow Co-Cultures

This Example demonstrates that anti-Jagged 4D11 inhibits the growth ofRPMI 8226 in human bone marrow co-cultures

In multiple myeloma, interaction between the myeloma cells and thestromal cells of the bone marrow is important for the survival andproliferation of myeloma cells and the development of the accompanyingosteolytic disease. Notch receptors and ligands are upregulated inmultiple myeloma. The ability of anti-Jagged 4D11 to inhibitproliferation of the multiple myeloma cell line RPMI 8226 was measuredin vitro in co-cultures of RPMI 8226 and human bone marrow aspirates.Human bone marrow was purchased from AllCells, LLC (Emeryville, Calif.).RPMI 8226 cells were labeled with CFSE as per manufacturer'sinstructions (Invitrogen, Carlsbad, Calif.). Briefly, bone marrow wasdiluted 2-fold in RPMI-1640, 10% FBS, and 2 mL were plated into thewells of a 6-well tissue culture dish. 50,000 CFSE-labeled RPMI 8226cells, in 1 ml RPMI1640, 10% FBS, were plated into wells containing bonemarrow. Test articles, i.e., anti-Jagged 4D11, anti-EGFR (antibody c225,cetuximab UCSF Pharmacy, manufactured and sold by Bristol-Myers Squibb,NY, N.Y.) or gamma secretase inhibitor BMS299897 (Sigma, St. Louis,Mo.), were added, and the cultures were incubated at 37° C. and 5% CO₂for five days. Following incubation, red blood cells were lysed, andlive cells were collected by centrifugation. The fluorescent intensityof the cells was measured by FACS.

Results are shown in FIG. 8. Reduced fluorescence, indicatingproliferation, was measured in the absence of any treatment or in thepresence of anti-EGFR. In contrast, both BMS299897 (GSI) and anti-Jagged4D11 inhibited the proliferation of CFSE-labeled RPMI 8226.

Example 9 An Anti-Jagged Antibody of the Embodiments InhibitsDevelopment of Fibrosis In Vitro

This Example demonstrates that anti-Jagged 4D11 inhibits the developmentof fibrosis in vitro.

The rat fibroblast cell line NRK-49F (ATCC, Manassas, Va.) responds tohuman transforming growth factor beta 1 (TGFβ1) by loss of cell-cellcontact, elevated production and deposition of collagen, and focidevelopment. NRK-F49 cells were plated at 50,000 cells/well, in a 6-welltissue culture dish and cultured overnight in RPMI-1640, 10% fetalbovine serum (FBS) to allow for attachment and monolayer formation.Medium was removed, and cells were washed twice with RPMI-1640, 1% heatinactivated FBS and cultured overnight in RPMI-1640, 1% heat inactivatedFBS. Following overnight incubation, anti-Jagged 4D11, TGFβ1, or thecombination of TGFβ1 and anti-Jagged 4D11 was added to cells in culture.Cells were cultured for 5 days and observed for a response to TGFβ1.

Results are illustrated in FIG. 9. Panel A shows that cultures ofNRK-F49 retain a characteristic monolayer when cultured in the presenceof 100 nM anti-Jagged 4D11. Panel B shows characteristic foci formationfor NRK-F49 cultured in the presence of 10 ng/mL TGFβ1. Panel C showsthat TGFb1-stimulated, fibrotic foci formation is completely inhibitedby 100 nM anti-Jagged 4D11 in cultures treated with 10 ng/mL TGFβ1.

Example 10 Activatable Anti-Jagged Antibody Masking Moieties

This Example describes identification of masking moieties (MM) to reducebinding of activatable anti-Jagged antibodies to their target.

Anti-Jagged 4D11 antibody and Fab were used to screen a random X₁₅peptide library with a total diversity of 2×10¹⁰, where X is any aminoacid, using a method similar to that described in PCT InternationalPublication Number WO 2010/081173, published 15 Jul. 2010. The screeningconsisted of one round of MACS and two rounds of FACS sorting. Theinitial MACS was done with protein-A Dynabeads (Invitrogen) and theanti-Jagged 4D11 antibody at a concentration of 250 nM. For MACS,approximately 1×10¹¹ cells were screened for binding and 6×10⁶ cellswere collected. StreptAvidin-PE was used as a fluorescent probe for theinitial FACS and anti-biotin-PE (Miltenyi) for the second FACS. Thebiotinylated anti-Jagged 4D11 antibody was used at a concentration of100 nM and 10 nM in the first and second round of FACS, respectively.The positive population from the second FACS round was verified to beinhibited by recombinant Jagged protein from binding to the anti-Jagged4D11 antibody and Fab. Individual peptide clones were identified bysequence analysis and subsequently verified for their ability to bindthe anti-Jagged 4D11 antibody and Fab, as shown in FIG. 10.

The sequences of the anti-Jagged masking moieties are listed in Table 9.

TABLE 9 Anti-Jagged masking moieties (MM) MM Amino Acid Sequence SEQ IDNO JS4874 PWCMQRQDFLRCPQP SEQ ID NO: 87 JS4879 QLGLPAYMCTFECLR SEQ IDNO: 88 JS4896 CNLWVSGGDCGGLQG SEQ ID NO: 89 JS4897 SCSLWTSGSCLPHSP SEQID NO: 90 JS4899 YCLQLPHYMQAMCGR SEQ ID NO: 91 JS4906 CFLYSCTDVSYWNNTSEQ ID NO: 92

Example 11 Affinity Maturation of Anti-Jagged Masking Moieties

This Example describes affinity maturation of anti-Jagged maskingmoieties.

The anti-Jagged binding peptides JS4874, JS4896, JS4899, and JS4906 wereaffinity matured by using a soft randomization approach. An eCPX celldisplay library, such as that described in PCT International PublicationNumber WO 2009/014726, was constructed with the nucleotide ratios shownin Table 10. Four libraries were constructed: 4874SR, 4896SR, 4899SR,and 4906SR. The final diversity for each library was approximately5×10⁹.

TABLE 10 Nucleotide ratios Original Base Ratio of Bases G G = 70%; T =8%; A = 11%; C = 11% T T = 70%; G = 8%; A = 11%; C = 11% A A = 80%; G =5%; T = 6%; C = 9% C C = 80%; G = 5%; T = 6%; A = 9%

Each affinity maturation library was screened separately. An initialMACS round was performed with a number of cells that provided greaterthan 100× oversampling of the library. All labeling was performed at 4°C. under constant gentle agitation. Cells were labeled with 25 nManti-Jagged Fab (library 4896SR) or 50 nM anti-Jagged antibody(libraries 4874SR, 4899SR, and 4906SR) and then bound to approximately500 μg streptavidin or protein-A labeled magnetic beads (DynabeadsInvitrogen). Beads were subsequently washed extensively with PBScontaining 0.5% BSA. Approximately 2×10⁶ to 2×10⁷ cells from eachlibrary were recovered from the initial MACS round.

Bacterial cells for all FACS rounds were labeled with biotinylatedanti-Jagged Fab. The secondary fluorescent label used was eitheranti-biotin-PE (Miltenyi) or streptavidin-PE (Invitrogen) depending onthe observed background binding of secondary label alone. For all FACSrounds, the brightest 2% of positive cells were sorted. For library4896SR, cells for FACS round 1 (F1) and FACS round 2 (F2) were labeledwith 2 nM and 1 nM Fab, respectively. For libraries 4874SR, 4899SR, and4906SR, cells for F1 were labeled with 100 nM Fab. For the 4874SRlibrary, cells for F2 were labeled with 1 nM Fab while cells forlibraries 4899SR and 4906SR were labeled with 10 nM Fab. Sequences fromthe FACS round 2 from each library are shown in Tables 11 through 14.

TABLE 11 Masking moiety sequences from FACS round 2 of library 4874SR4874SR M1F2 peptide sequences JF5336 PWCMQRQDYLRCPQP SEQ ID NO: 93

TABLE 12 Masking moiety sequences from FACS round 2 of library 4896SR4896SR M1F2 peptide sequences JF5411 CNLWISGGDCRGLAG SEQ ID NO: 94JF5416 CNLWVSGGDCRGVQG SEQ ID NO: 95 JF5421 CNLWVSGGDCRGLRG SEQ ID NO:96 JF5432 CNLWISGGDCRGLPG SEQ ID NO: 97 JF5436 CNLWVSGGDCRDAPW SEQ IDNO: 98 JF5439 CNLWVSGGDCRDLLG SEQ ID NO: 99 JF5424 CNLWVSGGDCRGLQG SEQID NO: 100 JS5340 CNLWLHGGDCRGWQG SEQ ID NO: 101 JS5342 CNIWLVGGDCRGWQGSEQ ID NO: 102 JS5345 CTTWFCGGDCGVMRG SEQ ID NO: 103 JS5347CNIWGPSVDCGALLG SEQ ID NO: 104 JS5358 CNIWVNGGDCRSFEG SEQ ID NO: 105

TABLE 13 Masking moiety sequences from FACS round 2 of library 4899SR4899SR M1F2 peptide sequences JF5366 YCLNLPRYMQDMCWA SEQ ID NO: 106JF5372 YCLALPHYMQADCAR SEQ ID NO: 107

TABLE 14 Masking moiety sequences from FACS round 2 of library 4906SR4906SR M1F2 peptide sequences JF5386 CFLYSCGDVSYWGSA SEQ ID NO: 108JF5387 CYLYSCTDSAFWNNR SEQ ID NO: 109 JF5388 CYLYSCNDVSYWSNT SEQ ID NO:110 JF5389 CFLYSCTDVSYW SEQ ID NO: 111 JF5390 CFLYSCTDVAYWNSA SEQ ID NO:112 JF5391 CFLYSCTDVSYWGDT SEQ ID NO: 113 JF5394 CFLYSCTDVSYWGNS SEQ IDNO: 114 JF5395 CFLYSCTDVAYWNNT SEQ ID NO: 115 JF5399 CFLYSCGDVSYWGNPGLSSEQ ID NO: 116 JF5402 CFLYSCTDVAYWSGL SEQ ID NO: 117 JF5404CYLYSCTDGSYWNST SEQ ID NO: 118 JF5405 CFLYSCSDVSYWGNI SEQ ID NO: 119JF5407 CFLYSCTDVAYW SEQ ID NO: 120 JF5409 CFLYSCTDVSYWGST SEQ ID NO: 121JF5410 CFLYSCTDVAYWGDT SEQ ID NO: 122

The binding of the parental masking moiety peptide JS4896 was comparedto the binding of masking moiety peptides selected from the 4896SRlibrary (clones JS5340, JS5342, JS5347, and JS5358). Cells containingthe indicated clones were analyzed on FACS at 3 different concentrationsof biotinylated anti-Jagged Fab, i.e., 1 nM, 10 nM, 100 nM.Streptavidin-PE was used as a secondary fluorescent label. Peptideexpression was quantified by labeling with yPet-MONA using techniquessimilar to those described in PCT WO 2007/027935. Results are shown inFIG. 11.

Example 12 Activatable Anti-Jagged Antibodies

This Example describes examples of activatable anti-Jagged antibodies ofthe disclosure.

Activatable anti-Jagged antibodies comprising an anti-Jagged maskingmoiety, a cleavable moiety, and an anti-Jagged antibody of thedisclosure were produced according to methods similar to those describedin PCT Publication Nos. WO 2009/025846 and WO 2010/081173. Qualitycontrol of the resultant activatable antibodies indicated that mostcomprised at least 95% monomer. The amino acid and nucleic acidsequences of several activatable anti-Jagged antibodies of thedisclosure are provided below.

The nucleic acid and amino acid sequences of the light chains (Lc) ofseveral activatable anti-Jagged antibodies comprising masking moietyJS5342 (also referred to herein as MM 5342 or 5342), a CM that can becleaved by at least one protease, and the light chain of AB 4D11 areshown below.

5342-1203-4D11 Lc Amino acid (SEQ ID NO: 132)QGQSGQCNIWLVGGDCRGWQGGSSGGSGGSGGTGRGPSWVGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 131)CAAGGCCAGTCTGGCCAATGCAATATTTGGCTCGTAGGTGGTGATTGCAGGGGCTGGCAGGGGGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTGGCCGTGGTCCAAGCTGGGTTGGCGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 5342-1204-4D11 Lc Amino acidsequence (SEQ ID NO: 134)QGQSGQCNIWLVGGDCRGWQGGSSGGSGGSGGLSGRSDNHGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEXFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 133)CAAGGCCAGTCTGGCCAGTGCAATATTTGGCTCGTAGGTGGTGATTGCAGGGGCTGGCAGGGGGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATAATCATGGCGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 5342-1214-4D11 Lc Amino acidsequence (SEQ ID NO: 136)QGQSGQCNIWLVGGDCRGWQGGSSGGSGGSGGSPLTGRSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLXIYAASSLQSGVPSRFSGSGSGTDFTLTISRLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 135)CAAGGCCAGTCTGGCCAGTGCAATATTTGGCTCGTAGGTGGTGATTGCAGGGGCTGGCAGGGGGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCACCACTGACTGGTCGTTCCGGTGGCGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 5342-PLGL-4D11 Lc Amino acidsequence (SEQ ID NO: 138)QGQSGQCNIWLVGGDCRGWQGGSSGGSGGSGGSGGGSPLGLGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 137)CAAGGCCAGTCTGGCCAGTGCAATATTTGGCTCGTAGGTGGTGATTGCAGGGGCTGGCAGGGGGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGGTGGAGGCTCGCCACTGGGCCTGGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

The nucleic acid and amino acid sequences of the light chain of a maskedantibody comprising masking moiety 5342, a noncleavable linker, and thelight chain of AB 4D11 are shown below:

5342-NSub-4D11 Lc Amino acid sequence (SEQ ID NO: 140)QGQSGQCNIWLVGGDCRGWQGGSSGGSSGSGGSGGGSGGGSGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 139)CAAGGCCAGTCTGGCCAGTGCAATATTTGGCTCGTAGGTGGTGATTGCAGGGGCTGGCAGGGGGGCTCGAGCGGTGGCAGCAGTGGCTCTGGTGGCTCAGGTGGAGGCTCGGGCGGTGGGAGCGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

The nucleic acid and amino acid sequences of several polypeptidesincluding MM 5342 and a CM that can be joined to an anti-Jagged antibodyof the disclosure using methods such as those described herein toproduce an activatable anti-Jagged antibody of the disclosure areprovided below:

5342-Cath.E Amino acid sequence (SEQ ID NO: 142)QGQSGQCNIWLVGGDCRGWQGGSSGGSGGSGGSAGFSLPAGGGS Nucleotide sequence (SEQ IDNO: 141) CAAGGCCAGTCTGGCCAGTGCAATATTTGGCTCGTAGGTGGTGATTGCAGGGGCTGGCAGGGGGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGCTGGCTTCTCCCTCCCCGCAGGTGGCGGTTCT 5342-MMP-14 (SEQ ID NO: 144)QGQSGQCNIWLVGGDCRGWQGGSSGGSGGSGSLAPLGLQRRGGS Nucleotide sequence (SEQ IDNO: 143) CAAGGCCAGTCTGGCCAGTGCAATATTTGGCTCGTAGGTGGTGATTGCAGGGGCTGGCAGGGGGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTAGCCTGGCACCTCTGGGTCTGCAACGCCGTGGCGGTTCT 5342-panMMP Amino acid sequence (SEQID NO: 146) QGQSGQCNIWLVGGDCRGWQGGSSGGSGGSGGSGGPLGVRGGGS Nucleotidesequence (SEQ ID NO: 145)CAAGGCCAGTCTGGCCAGTGCAATATTTGGCTCGTAGGTGGTGATTGCAGGGGCTGGCAGGGGGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGGTGGACCTTTGGGAGTCAGAGGTGGCGGTTCT

The Cathepsin E (Cath E), MMP-14, and panMMP substrates (CMs) used toproduce these polypeptides have been reported in the literature:Cruz-Monserrate Z et al., 2011, Gut, doi:10.1136/gutjnl-2011-300544;Abeer J et al., 2011, Chem. Biol. 18, 392-401; Zhu L et al., 2011,Theranostics 1, 18-27.

Examples of antibodies to which such MM and CM containing polypeptidescan be joined include anti-Jagged antibody 4D11 or a variant thereof.The amino acid and nucleic acid sequences of the heavy chain of the 4D11variant, referred to as 4D11 Hc QΔH, are provided below:

4D11 Hc QAH Amino acid (SEQ ID NO: 148)EVHLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK Nucleotide(SEQ ID NO: 147) GAGGTGCACCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAAGTATTGACCCGGAAGGTCGGCAGACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACATCGGCGGCAGGTCGGCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

Example 13 In Vitro Characterization of Activatable Anti-JaggedAntibodies

This Example describes the ability of a masking moiety of the disclosureto reduce the ability of activatable anti-Jagged antibodies comprisingsuch a masking moiety to bind to a Jagged target. This Example alsodescribes proteolytic activation of such activatable antibodies.

The abilities of activatable antibodies 5342-1203-4D11, 5342-1204-4D11,5342-1214-4D11, and 5342-PLGL-4D11, as well as masked antibody5342-NSub-4D11, to bind to a human Jagged 1 target were compared to theability of anti-Jagged antibody 4D11 to bind to the same target in an invitro binding assay as described herein. The ability of MM 5342 toinhibit such target binding is demonstrated in FIG. 12 and Table 15.

TABLE 15 Comparison of Jagged target binding by anti-Jagged antibody4D11 and by a masked antibody and activatable antibodies thereof. Foldmasking calculated as (K_(D) apparent for activatable antibody/K_(D)apparent for antibody 4D11). Fold masking Activatable antibody5342-1203-4D11 52.7 5342-1204-4D11 127.0 5342-1214-4D11 64.75342-PLGL-4D11 131.7 Masked antibody 5342-NSub-4D11 31.2

Activatable anti-Jagged antibodies 5342-1203-4D11, 5342-1204-4D11,5342-1214-4D11, and 5342-PLGL-4D11, as well as masked antibody5342-NSub-4D11, were assessed for their abilities to be cleaved byproteases. Briefly, 250 ng of activatable antibody was digested by 1 uMuPA or 387 nM MMP-2 for 24 hours at 37° C. in the appropriate buffer(for uPA: 0.1M Tris pH 8.0 in HBSS; for MMP-2: TCNB (50 mM Tris, 150 mMNaCl, 0.05% Brij, 10 mM CaCl2, pH 9.5)). The digested material wassubsequently analyzed by SDS-PAGE and western blotting, using goatanti-human IgG Fab′2 HRP as a detection agent. FIG. 13 demonstrates thatproteolytic digestion yielded a protein with a mobility similar to thatof the parental (i.e., antibody 4D11) light chain, indicating that therespective activatable antibodies were cleaved with uPA or MMP-2proteases, respectively.

Example 14 In Vivo Characterization of Activatable Anti-JaggedAntibodies

This Example describes the in vivo efficacy and safety of activatableanti-Jagged antibodies of the disclosure in a mouse BxPC3 tumor model.

Activatable anti-Jagged antibodies 5342-1203-4D11, 5342-1204-4D11,5342-1214-4D11, and 5342-PLGL-4D11, as well as masked antibody5342-NSub-4D11, were tested for their abilities to reduce the growth ofBxPC3 xenograft tumors implanted into mice, using a method similar tothat described herein. Also tested was the ability of these activatableand masked antibodies to reduce weight loss in such a tumor modelcompared to weight loss caused by anti-Jagged antibody 4D11. The groups,doses, dosing route, and dosing schedule are set forth in Table 16.Efficacy results (reduction in tumor size) are shown in FIG. 14. Safetyresults (reduction in weight loss) are shown in FIG. 15. Serumconcentrations of mouse thymic stromal lymphopoietin (TSLP) are shown inFIG. 16A.

TABLE 16 Groups and doses for the activatable anti-Jagged antibody BxPC3efficacy study Dose Dosing Group N Treatment (mg/kg) Route Schedule 1 8PBS NA i.p. q7dx 4 2 8 4D11 6.7 mg/kg  i.p. q7dx 4 3 8 4D11 20 mg/kgi.p. q7dx 4 4 8 5342-NSub-4D11 20 mg/kg i.p. q7dx 4 5 8 5342-1203-4D1120 mg/kg i.p. q7dx 4 6 8 5342-1204-4D11 20 mg/kg i.p. q7dx 4 7 85342-1214-4D11 20 mg/kg i.p. q7dx 4 8 8 5342-PLGL-4D11 20 mg/kg i.p.q7dx 4

FIG. 14, which plots tumor volume versus number of days post initialdose, demonstrates that activatable anti-Jagged antibodies inhibited thegrowth of BxPC-3 xenograft tumors in mice, as did anti-Jagged antibody4D11 (parental antibody).

FIG. 15 compares weight loss of mice administered activatableanti-Jagged antibodies, masked antibody, or parental antibody. While theanimals dosed with parental antibody 4D11 showed significant weightloss, animals dosed with an activatable anti-Jagged antibody showed nosignificant weight loss.

Serum concentrations of mouse thymic stromal lymphopoietin (TSLP) weremeasured as described herein. The serum levels of mouse TSLP wasquantified for individual mice before each dose and 10 days after thefinal dose from each group and averaged to generate FIG. 16A. FIG. 16Bdepicts a time course of TSLP serum concentrations for anti-Jaggedantibody 4D11 and activatable anti-Jagged antibody 5342-1204-4D11. Serummouse TSLP is elevated in the parenteral anti-Jagged antibody 4D11groups compared to serum mouse TSLP levels in the groups administeredactivatable anti-Jagged antibodies.

Example 15 Pharmacokinetic Data of Activatable Anti-Jagged Antibodies

This Example compares the pharmacokinetics of anti-Jagged parental andactivatable antibodies in the sera of mice administered such antibodies.

Single dose pharmacokinetics in non-tumor-bearing female Balb/c nudemice administered anti-Jagged antibody 4D11 or activatable anti-Jaggedantibody 5342-1204-4D11 were evaluated. The mice were dosed as outlinedin Table 17. Cohorts of five mice were bled in rotation at 0.5, 3, 8,24, 72, 168, and 240 hrs. Plasma samples were analyzed for hIgG contentusing an anti-hFc capture with subsequent detection with an anti-hIgGFab′2 HRP conjugate.

TABLE 17 Groups and doses for the study comparing pharmacokinetics ofparental and activatable anti-Jagged antibodies. Group Count TreatmentDose (mg/kg) Route 1 20 4D11 6.7 mg/kg IP 2 20 5342-1204-4D11 6.7 mg/kgIP

FIG. 17 compares the average human IgG levels over time in the sera ofmice following intraperitoneal administration of activatable anti-Jaggedantibody 5342-1204-4D11 (also referred to herein as 5342-1204) oranti-Jagged antibody 4D11. Mice administered anti-Jagged antibody 4D11intravenously showed similar human IgG levels over time as miceadministered the same antibody intraperitoneally.

Table 18 provides a preliminary noncompartmental analysis through day 7.The data were analyzed using Phoenix WinNonlin version 6.3, sparsesampling mode.

TABLE 18 Anti-Jagged pharmacokinetics (PK) study preliminarynoncompartmental analysis through day 7. Half life Tmax Cmax AUClastSE_AUClast Group hr hr ug/mL hr * ug/mL hr * ug/mL 4D11 28 3 118 7,431432 5342-1204 187 8 81 11,613 690

Example 16 Additional Maturation of Anti-Jagged Masking Moieties

This Example describes the production of additional anti-Jagged maskingmoieties of the disclosure.

To further affinity mature masking moiety peptide family JS4896 (alsoreferred to herein as MM 4896 or 4896), the sequences from the SRlibrary screens described above were used to design four directedaffinity maturation libraries. An eCPX cell display library, such asthat described in PCT International Publication Number WO 2009/014726,was constructed with the nucleotide sequence shown in Table 19. Thefinal diversity for each library was approximately 5×10⁹ cells.

TABLE 19 Peptide family 4896-directed library designs Name Librarydesign (nucleotides) 1517/1519 TGCAATMTKTGGVBCNNKGGTGGTGATTGCCGCGGGTGGNNKNNKNNKNNKNNK (SEQ ID NO: 149) 1518/1521NNKNNKNNKNNKTGCAATMTKTGGVBCNNKGGTGGT GATTGCCGCGGGTGGNNK (SEQ ID NO: 150)1559 TGCAATMTKTGGVBCNNKGGTGGTGATTGCCGCNNKN NKNNKNNKNNK (SEQ ID NO: 151)1561 NNKNNKNNKNNKTGCAATMTKTGGVBCNNKGGTG GTGATTGCCGCNNK (SEQ ID NO: 152)

Libraries 1517/1519 and 1518/1521

Each affinity maturation library was screened separately but in the samemanner. An initial MACS round was performed with a number of cells thatprovided greater than 100× oversampling of the library. All labeling wasperformed at 4° C. under constant gentle agitation. Cells were labeledwith 25 nM Fab 4D11 labeled with biotin. Cells that bound to the Fabwere captured using streptavidin-labeled magnetic beads (Dynabeads,Invitrogen). Beads were subsequently washed extensively with PBScontaining 0.5% BSA. Approximately 1×10⁶ cells from each library wererecovered from the initial MACS round.

Bacterial cells for all FACS rounds were labeled with DyLight-488labeled anti-Jagged Fab 4D11 (i.e., the Fab of anti-Jagged IgG antibody4D11). For all FACS rounds, the brightest 0.1% to 0.2% of positive cellswere sorted. Cells for FACS round 1 (F1) and FACS round 2 (F2) werelabeled with 1 nM and 100 pM Fab 4D11, respectively. For FACS rounds 3and 4, cells were labeled with 1 nM DyLight-labeled anti-Jagged Fab4D11, resuspended in 500 μl PBS and incubated at 37° C. for between 5and 10 minutes before sorting. The clones that were sorted in FACS round4 were sequenced and the results are shown in Tables 20 and 21.

TABLE 20 Anti-Jagged masking moieties (MM) 4896 directed library1517/1519 peptide sequences JS5872 GCNIWLNGGDCRGWVDPLQG (SEQ ID NO: 153)JS5877 GCNIWLVGGDCRGWIGDTNG (SEQ ID NO: 154) JS5885 GCNIWLVGGDCRGWIEDSNG(SEQ ID NO: 155) JS5887 GCNIWANGGDCRGWIDNIDG (SEQ ID NO: 156) JS5937GCNIWLVGGDCRGWLGEAVG (SEQ ID NO: 157) JS5954 GCNIWLVGGDCRGWLEEAVG (SEQID NO: 158)

TABLE 21 Anti-Jagged masking moieties (MM) 4896 directed library1518/1521 peptide sequences JS5892 GGPALCNIWLNGGDCRGWSG (SEQ ID NO: 159)JS5893 GAPVFCNIWLNGGDCRGWMG (SEQ ID NO: 160) JS5894 GQQQWCNIWINGGDCRGWNG(SEQ ID NO: 161) JS5899 GKSEFCNIWLNGGDCRGWIG (SEQ ID NO: 162) JS5902GTPGGCNIWANGGDCRGWEG (SEQ ID NO: 163) JS5908 GASQYCNLWINGGDCRGWRG (SEQID NO: 164)

Individual clones were evaluated by FACS for Fab binding. An example isshown in FIG. 18. The clones expressing masking moieties from thedirected libraries (MM 5872, 5877, 5885, and 5887) bound the Fab 4D11better than a single clone expressing MM 5342 from the SR library sorts.

Libraries 1559 and 1561

Affinity maturation libraries 1559 and 1561 were screened separately butin the same manner. An initial MACS round was performed as above butwith 50 nM Fab 4D11 labeled with biotin. Approximately 1×10⁶ cells fromeach library were recovered from the initial MACS round.

Bacterial cells for all FACS rounds were labeled with DyLight-488labeled anti-Jagged Fab 4D11. For all FACS rounds, the brightest 0.2% ofpositive cells were sorted. Cells for FACS round 1 (F1) and FACS round 2(F2) were labeled with 1 nM Fab 4D11. For FACS rounds 3 and 4, cellswere labeled with 1 nM DyLight-labeled anti-Jagged Fab 4D11, resuspendedin 500 μl PBS and incubated at 37° C. for between 5 and 10 minutesbefore sorting. The clones that were sorted in FACS round 4 weresequenced, and the results are shown in Tables 22 and 23.

TABLE 22 Anti-Jagged masking moieties (MM) 4896-directed library 1559peptide sequences JS6094 GCNIWLVGGDCRPWVEGG (SEQ ID NO: 165) JS6095GCNIWAVGGDCRPFVDGG (SEQ ID NO: 166) JS6097 GCNIWLNGGDCRAWVDTG (SEQ IDNO: 167) JS6098 GCNIWIVGGDCRPFINDG (SEQ ID NO: 168) JS6099GCNIWLNGGDCRPVVFGG (SEQ ID NO: 169) JS6101 GCNIWLSGGDCRMFMNEG (SEQ IDNO: 170) JS6104 GCNIWVNGGDCRSFVYSG (SEQ ID NO: 171) JS6108GCNIWLNGGDCRGWEASG (SEQ ID NO: 172) JS6110 GCNIWAHGGDCRGFIEPG (SEQ IDNO: 173) JS6112 GCNIWLNGGDCRTFVASG (SEQ ID NO: 174) JS6116GCNIWAHGGDCRGFIEPG (SEQ ID NO: 175)

TABLE 23 Anti-Jagged masking moieties (MM) 4896 directed library 1561peptide sequences JS6118 GFLENCNIWLNGGDCRTG (SEQ ID NO: 176) JS6119GIYENCNIWLNGGDCRMG (SEQ ID NO: 177) JS6126 GIPDNCNIWINGGDCRYG (SEQ IDNO: 178)

Example 17 Additional Activatable Anti-Jagged Antibodies

This Example describes additional examples of activatable anti-Jaggedantibodies of the disclosure.

The nucleic acid and amino acid sequences of several polypeptidesincluding masking moiety JS5894 (also referred to herein as MM 5894 or5894) and a CM that can be joined to an anti-Jagged antibody of thedisclosure using methods such as those described herein to produce anactivatable anti-Jagged antibody of the disclosure are provided below:

Mask 5894 that also includes a 6-amino acid N-terminal spacer Amino acidsequence (SEQ ID NO: 180) QGQSGQGQQQWCNIWINGGDCRGWNG Nucleic acidsequence (SEQ ID NO: 179)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGT 5894-1203 Amino acid sequence (SEQ ID NO:182) QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGTGRGPSWVGGGS Nucleotidesequence (SEQ ID NO: 181)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTGGCCGTGGTCCAAGCTGGGTTGGCGGCGGTTCT 5894-1203-4D11 Lc Aminoacid sequence (SEQ ID NO: 263)QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGTGRGPSWVGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 264)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTACTGGCCGTGGTCCAAGCTGGGTTGGCGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 5894-1204 Amino acid sequence(SEQ ID NO: 184) QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGLSGRSDNHGGGSNucleotide sequence (SEQ ID NO: 183)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATAATCATGGCGGCGGTTCT 5894-1204-4D11 Lc Aminoacid sequence (SEQ ID NO: 265)QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGLSGRSDNHGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 266)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATAATCATGGCGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 5894-1214 Amino acid sequence(SEQ ID NO: 186) QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGSPLTGRSGGGGSNucleotide sequence (SEQ ID NO: 185)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCACCACTGACTGGTCGTTCCGGTGGCGGCGGTTCT 5894-1214-4D11 Lc Aminoacid sequence (SEQ ID NO: 267)QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGSPLTGRSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 268)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCACCACTGACTGGTCGTTCCGGTGGCGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 5894-PLGL Amino acid sequence(SEQ ID NO: 188) QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGSGGGSPLGLGGSNucleotide sequence (SEQ ID NO: 187)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGGTGGAGGCTCGCCACTGGGCCTGGGCGGTTCT 5894-PLGL-4D11 Lc Aminoacid sequence (SEQ ID NO: 269)QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGSGGGSPLGLGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 270)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGGTGGAGGCTCGCCACTGGGCCTGGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 5894-Cath.E Amino acid sequence(SEQ ID NO: 190) QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGSAGFSLPAGGGSNucleotide sequence (SEQ ID NO: 189)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGCTGGCTTCTCCCTCCCCGCAGGTGGCGGTTCT 5894-Cath.E-4D11 LcAmino acid sequence (SEQ ID NO: 271)QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGSAGFSLPAGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 272)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGCTGGCTTCTCCCTCCCCGCAGGTGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 5894-MMP-14 Amino acid sequence(SEQ ID NO: 192) QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGSLAPLGLQRRGGSNucleotide sequence (SEQ ID NO: 191)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTAGCCTGGCACCTCTGGGTCTGCAACGCCGTGGCGGTTCT 5894-MMP-14-4D11 LcAmino acid sequence (SEQ ID NO: 273)QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGSLAPLGLQRRGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 274)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTAGCCTGGCACCTCTGGGTCTGCAACGCCGTGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 5894-panMMP Amino acid sequence(SEQ ID NO: 194) QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGSGGPLGVRGGGSNucleotide sequence (SEQ ID NO: 193)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGGTGGACCTTTGGGAGTCAGAGGTGGCGGTTCT 5894-panMMP-4D11 LcAmino acid sequence (SEQ ID NO: 275)QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGSGGPLGVRGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 276)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGGTGGACCTTTGGGAGTCAGAGGTGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

The nucleic acid and amino acid sequences of a polypeptide includingmasking moiety JS 5894 and a noncleavable linker that can be joined toan anti-Jagged antibody using methods such as those described herein toform a masked antibody are provided below:

5894-NSUB Amino acid sequence (SEQ ID NO: 196)QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGSGGGSGGGSGGS Nucleotide sequence(SEQ ID NO: 195) CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGGTGGAGGCTCGGGCGGTGGGAGCGGCGGTTCT 5894-NSUB-4D11 Lc Aminoacid sequence (SEQ ID NO: 278)QGQSGQGQQQWCNIWINGGDCRGWNGGSSGGSGGSGGSGGGSGGGSGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECNucleotide sequence (SEQ ID NO: 279)CAAGGCCAGTCTGGCCAGGGTCAGCAGCAGTGGTGCAATATTTGGATCAATGGTGGTGATTGCCGCGGGTGGAATGGTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGGTGGAGGCTCGGGCGGTGGGAGCGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

Examples of antibodies to which such MM and CM containing polypeptidescan be joined include anti-Jagged antibody 4D11 or variants thereof,such as the 4D11 QAH variant described above.

Example 18 In Situ Imaging of Activatable Anti-Jagged Antibodies

The present Example describes the use of in situ imaging of theactivation and binding of an activatable anti-Jagged antibody of thedisclosure. The results indicate that activatable anti-Jagged antibodiesof the disclosure can be activated by proteases expressed by a tissueand bind Jagged targets on that tissue.

In situ imaging of activatable antibodies represents a unique approachto characterize protease activity in cells and tissue. This technologyenables validation of activatable antibody activation and binding to atarget in histological sections of cells and tissues expressingproteases capable of cleaving the activatable antibody. A schematic ofsuch an in situ approach is presented in FIG. 19.

In situ imaging of the activation and binding of an activatableanti-Jagged antibody (also referred to herein as in situ imaging) by acell or tissue capable of cleaving the activatable antibody at a siteco-localized with the target recognized by the activated antibody wasconducted as follows: Frozen tissue sections were laid over glassslides. A solution containing labeled activatable anti-Jagged antibodies(labeled, e.g., with a fluorescent tag) was applied on the tissue andincubated, e.g., for 1 hour at room temperature (about 22-24° C.) in anincubation buffer of 50 mM Tris-HCl buffer pH 7.4, containing 150 mMNaCl, 100 μM ZnCl₂, 5 mM CaCl₂ and 0.05% Tween 20; activatable antibodyat a concentration of about 1 μg/ml. The tissue was then extensivelywashed to remove non-bound material and detectable label was measured.For example, when a fluorescent tag was used, the tissue was submittedto fluorescent microscopy. Detection of activated antibody on the tissueindicated that the tissue expressed proteases that cleaved theactivatable antibody and also expressed Jagged targets to which theactivated antibody bound.

The abilities of activatable anti-Jagged antibodies 5342-1204-4D11 and5342-PLGL-4D11 to be activated and to bind BxPC3 xenograft tumor tissuewere evaluated using in situ imaging. The activatable antibodies werelabeled with Alexa Fluor® 680 (Invitrogen) to produce labeledactivatable antibodies 5342-1204-4D11-AF680 and 5342-PLGL-4D11-AF680,also referred to herein as 1204-4D11-AF680 and PLGL-4D11-AF680,respectively. Also tested was labeled anti-Jagged parental antibody4D11-AF680. Each of 4D11-AF680, 1204-4D11-AF680 and PLGL-4D11-AF680 wasincubated with a frozen BxPC3 xenograft tumor tissue sample as describedabove. The results are shown on FIG. 20, panels A, B, and C,respectively. The red fluorescent tissue images demonstrate binding of4D11 antibody and of 4D 11 antibodies activated by tissue-derivedproteolytic cleavage of the respectively activatable antibodies toJagged. Panels D, E, and F represent the fluorescent images obtainedafter incubation of 4D11-AF680, 1204-4D11-AF680 and PLGL-4D11-AF680 withfrozen BxPC3 xenograft tumor tissue pre-treated with a 1:100 dilution ofbroad spectrum protease inhibitor cocktail set III (Catalog No. 539134,EMD Millipore) and 50 mM EDTA. Reduced red fluorescence in panels E andF indicates that the binding of activatable antibodies 1204-4D11-AF680and PLGL-4D11-AF680 seen in panels B and C was effected by cleavage ofthe activatable antibodies by tissue-derived proteases; the proteaseinhibitor cocktail inhibited such proteolysis. Blue staining representsDAPI nuclear staining. Binding of anti-Jagged parental antibody 4D11 orof activatable anti-Jagged antibodies 5342-1204-4D11 and 5342-PLGL-4D11to frozen BxPC3 xenograft tumor tissue was inhibited by pre-treatingsuch tissue with unlabeled anti-Jagged parental antibody 4D11 or bypre-treating such tissue with Jagged 1, Jagged 2, or a combinationthereof.

Activation of activatable anti-Jagged antibodies 5342-1204-4D11 and5342-PLGL-4D11 were also evaluated by in situ imaging of humanpancreatic cancer tissue. Each of 4D11-AF680 4D11), 1204-4D11-AF680(1204) and PLGL-4D11-AF680 (PLGL) was incubated with a frozen tissuesample isolated from a human patient with pancreatic cancer. The resultsare shown on FIG. 21, panels in column 1, rows 1, 2, and 3,respectively. The panels in Columns 2, 3, and 4, respectively, representthe fluorescent images obtained after incubation of 4D11-AF680,1204-4D11-AF680 and PLGL-4D11-AF680 with frozen pancreatic cancerpatient tissue pre-treated with 10 μg/ml of antibody A11 (A11 is anantibody that specifically binds to the active site of the MT-SP1protease, also known as matriptase) (FIG. 21, column 2) with 50 μM ofbroad spectrum MMP inhibitor Galardin (Calbiochem, Millipore) (FIG. 21,column 3) or with a 1:100 dilution of broad spectrum protease inhibitorcocktail set III (Cat. No. 539134, EMD Millipore) and 50 μM broadspectrum MMP inhibitor Galardin (Calbiochem, Millipore) (FIG. 21, column4). Blue staining represents DAPI nuclear staining. The results suggestthat the pancreatic tissue sample produces active matriptase andmetalloprotease, the presence of which effects cleavage of respectiveactivatable antibody cleavable moieties, thereby releasing the maskingmoiety and enabling stable binding of the activated antibody to Jaggedtargets on the tissue.

Example 19 In Vivo Imaging of an Anti-Jagged Antibody

The present Example describes the in vivo imaging of an anti-Jaggedantibody of the disclosure.

Anti-Jagged antibody 4D11 was labeled with Alexa Fluor® 750 and purifiedfrom unconjugated dye using 40 kDa Thermo Scientific Zeba Spin DesaltingColumns. A group of three mice bearing BxPC3-luc human pancreatic cancertumors with tumor volumes of approximately 400-600 mm³ were administeredintraperitoneally (i.p.) a single 10-mg/kg dose of Alexa Fluor®750-labeled 4D11 antibodies (n=3). Mice were anesthetized withisoflurane and imaged for 750 nm near-infrared (NIR) fluorescence priorto injection and at 24 h, 48 h, and 72 h post injection using theCaliper IVIS SpectrumCT imaging system (Caliper, Perkin Elmer, HopkintonMass.). Mice were euthanized after the last imaging time-point. FIG. 22Aprovides a representation of the labeled 4D11 antibody fluorescencesignal 48 hours post-injection in the BxPC3 tumor xenograft mouse model.

In vivo imaging data were normalized and analyzed using Living Image®4.1 software. For quantitative comparison, the regions of interest (ROI)were drawn over tumor (T) and normal tissue (N). The fluorescencesignal, quantified as Average Radiant Efficiency (photons×cm-2×s-1), foreach area was measured. The ratio of the signal in the tumor ROIcompared to the normal tissue ROI (T/N) was calculated to provide ameasure of the rate of anti-Jagged antibody 4D11 accumulation in thetumor versus normal tissue. FIG. 22B provides a graph showing the meanT/N ratio of average radiant efficacy for the antibody 4D11 dose group±SD.

Example 20 In Situ Imaging of Activatable Anti-Jagged Antibodies

The present Example describes the use of in situ imaging to screenpancreatic cancer xenograft tumor tissue and human pancreatic cancertissue for the activation and binding of an activatable anti-Jaggedantibody. The results indicate that activatable anti-Jagged antibodiesof the disclosure can be activated by proteases expressed by suchtissues and bind Jagged targets on such tissues.

BxPC3 tumor samples and human pancreatic cancer tissue samples wereprofiled for Jagged and MT-SP1 expression by 1 hour treatment of frozentissue with labeled anti-Jagged antibody 4D11 and anti-matriptase A11antibody at 1 μg/ml and 5 μg/ml concentrations, respectively. Theresults are shown in Table 24, columns 2 and 3, respectively.

In addition, the abilities of activatable anti-Jagged antibodies5342-1204-4D11 and 5342-PLGL-4D11 to be activated and to bind BxPC3xenograft and human pancreatic cancer tissues were evaluated using insitu imaging. The activatable antibodies were labeled with Alexa Fluor®680 (Invitrogen) as described above (Example 18). These labeledactivatable antibodies, i.e., 5342-1204-4D11-AF680 (also referred toherein as 1204-4D11-AF680) and 5342-PLGL-4D11-AF680 (also referred toherein as PLGL-4D11-AF680), were incubated with frozen BxPC3 xenografttissue or with human pancreatic cancer tissue samples isolated from fourpatients according to the protocol of in situ imaging described above(Example 18). Table 24 summarizes the results demonstrating the abilityof BxPC3 tumor and pancreatic cancer patients' tissue samples toactivate and bind activated activatable anti-Jagged antibodies. In Table24, the IHC staining that measured the amount of anti-Jagged antibody4D11 or anti-matriptase antibody A11 binding to the tissue samples(columns 2 and 3) was scored from 0 to 3+: 0, no staining; 1+, weakstaining; 2+, moderate staining; and 3+, strong staining. The in situimaging staining (columns 4 and 5) scoring is based on comparison with4D11 antibody staining and defined as follows: 0, no staining; 1+, weakstaining as compared to parental antibody; 2+, moderate staining ascompared to parental antibody; and 3+, analogous staining to parentalantibody. The BxPC3 results are also shown in FIG. 20.

TABLE 24 Screening for Jagged and MT-SP1 expression and in situ imagingof activatable anti-Jagged antibodies in BxPC3 xenograft and humanpancreatic cancer tissues. IHC in situ imaging Specimen # 4D11 A114D11-1204-AF680 4D11-PLGL-AF680 BxPC3 ++ ++ +++ +++ 5587 ++ + ++ +++5617 +++ ++ +++ ++ 5623 +++ ++ ++ + 5631 ++ − ++ +

Example 21 In Vitro Characterization of an Activatable AntibodyConjugated to an Agent

This Example describes the ability of an activatable antibody-agentconjugate of the disclosure to inhibit proliferation of BxPC3 cells inculture.

Activatable anti-Jagged antibody 5342-1204-4D11, anti-Jagged antibody4D11, and Rituxan were each conjugated to monomethylauristatin E (MMAE),a synthetic anti-mitotic tubulin polymerization inhibitor, to generateactivatable antibody-agent conjugate 5342-1204-4D11-MMAE andantibody-agent conjugates 4D11-MMAE and Rituxan-MMAE.

The abilities of the following compounds to inhibit BxPC3 cellproliferation in cell culture were determined: Activatable anti-Jaggedantibody-agent conjugate 5342-1204-4D11-MMAE; activatable anti-Jaggedantibody-agent conjugate 5342-1204-4D11-MMAE activated by uPA;activatable anti-Jagged antibody 5342-1204-4D11; activatable anti-Jaggedantibody 5342-1204-4D11 activated by uPA; anti-Jagged antibody 4D11;anti-Jagged antibody-agent conjugate 4D11-MMAE; Rituxan; andRituxan-MMAE. Activation of activatable antibody and activatableantibody-agent conjugate was effected by digestion overnight at 37° C.with active site-titrated uPA (500 nM) in Tris pH 8.5; activation wasmeasured by CE analysis (LabChip GXII). uPA-activated activatableantibody and uPA-activated activatable antibody-agent conjugate werepurified using protein A and then stored at 4° C. prior to the study.

The human pancreatic cancer cell line BxPC-3 was obtained from ATCC.BxPC-3 cells were grown in complete media (RPMI-1640 supplemented with10% fetal bovine serum) at 37° C. in an atmosphere of 5% CO2 in air.BxPC-3 cells were harvested during the logarithmic growth period,resuspended in complete medium, and plated at a density of 5000 cellsper well in a 96-well white wall chimney plate. Following overnightincubation, a 10-point 1:3 serial dilution, starting at 10 ug/ml andending in 0 of each compound was added to cells in culture inreplicates. Cells were cultured for 3 days and cell viability wasmeasured using CellTiterGlo (Promega) following manufacturer's protocoland a luminometer (Tecan). Data were analyzed using Prism GraphPad. Theresults are shown in FIG. 24.

Example 22 In Vivo Efficacy and Safety of an Activatable Antibody-AgentConjugate

This Example describes the ability of an activatable antibody-agentconjugate of the disclosure to reduce the growth of BxPC3 xenografttumors in vivo.

Activatable anti-Jagged antibodies and activatable anti-Jaggedantibody-agent conjugates were tested for their ability to reduce thegrowth of BxPC3 xenograft tumors, using a method similar to thatdescribed above, using the compounds, groups, and doses set forth inTable 25.

TABLE 25 Groups and Dosing Regimens Dose Dosing Group N Treatment(mg/kg) Route Schedule 1 8 PBS NA i.v. q7dx 4 2 8 4D11 6.7 mg/kg i.v.q7dx 4 3 8 4D11-MMAE 6.7 mg/kg i.v. q7dx 4 4 8 5342-1204-4D11 6.7 mg/kgi.v. q7dx 4 5 8 5342-1204-4D11- 6.7 mg/kg i.v. q7dx 4 MMAE

FIG. 25, which plots tumor volume versus number of days post initialdose, demonstrates that both anti-Jagged antibody 4D11-MMAE andactivatable anti-Jagged antibody 5342-1204-4D11-MMAE inhibited BxPC-3xenograft tumor growth more effectively than their unconjugatedcounterparts.

FIG. 26 shows the weight loss of the various groups. While the animalsdosed with anti-Jagged antibody 4D11 or anti-Jagged antibody-MMAE showedsignificant weight loss, animals dosed with activatable anti-Jaggedantibody 5342-1204-4D11 or activatable anti-Jagged antibody-agentconjugate 5342-1204-4D11-MMAE did not show significant weight loss.

Example 23 In Vivo Efficacy and Safety of Anti-Jagged Antibodies andActivatable Antibodies in Combination with Gemcitabine in the BxPC3Tumor Model

Anti-Jagged activatable antibodies were tested for their ability toreduce the growth of BxPC3 xenograft tumors, using a method similar tothat described above, using the antibodies, activatable antibodies,groups, and doses set forth in Table 26.

TABLE 26 Groups and doses for the anti-Jagged activatable antibody BxPC3efficacy study Dose Dosing Group N Treatment (mg/kg) Route Schedule 1 8PBS NA i.p q7dx 4 2 8 Gemcitabine 100 mg/kg i.p. q7dx 4 3 8 2 mg/kg4D11 + 2 mg/kg + i.p. q7dx 4 Gemcitabine 100 mg/kg 4 8 6.7 mg/kg 4D11 +6.7 mg/kg + i.p. q7dx 4 Gemcitabine 100 5 8 20.0 mg/kg 4D11 + 20 mg/kg +i.p. q7dx 4 Gemcitabine 100 mg/kg 6 8 2 mg/kg 5342- 2 mg/kg + i.p. q7dx4 1204-4D11 + 100 mg/kg Gemcitabine 7 8 6.7 mg/kg 5342- 6.7 mg/kg + i.p.q7dx 4 1204-4D11 + 100 mg/kg Gemcitabine 8 8 20.0 mg/kg 5342- 20 mg/kg +i.p. q7dx 4 1204-4D11 + 100 mg/kg Gemcitabine

FIG. 29, which plots tumor volume versus number of days post tumorinoculation, demonstrates that the anti-Jagged activatable antibody5342-1204-4D11 in combination with Gemcitabine inhibits the growth ofBxPC-3 xenograft tumors. The first dose was given on day 30. FIG. 30indicates the weight loss for the Gemcitabine alone group and for thegroup administered antibody in combination with Gemcitabine. While theanimals dosed with higher doses of antibody and Gemcitabine showedsignificant weight loss, animals dosed with activatable antibody andGemcitabine showed no weight loss over that of Gemcitabine alone. Theantibody at 20 mg/kg was not tolerated when given in combination withGemcitabine, resulting in the sacrifice of that group at day 49 due tobody weight loss. However, the activatable antibody at 20 mg/kg incombination with Gemcitabine was tolerated and showed equivalentefficacy to that of the antibody at 6.7 and 20 mg/kg in combination withGemcitabine. The serum concentration of mouse thymic stromallymphopoietin (TSLP) was measured as described above. The serum levelsof mouse TSLP (mTSLP) were quantified for individual mice before thesecond dose. Only the 20 mg/kg antibody in combination with Gemcitabinegroup showed elevated serum mTSLP, as indicated in FIG. 31.

Example 24 In Vivo Efficacy of an Anti-Jagged Antibody in Prostate andMammary Tumor Models

The efficacy of the anti-Jagged 4D11 antibody was evaluated inautocthonous tumor models for prostate and mammary cancer. These modelsmimic the human condition as the produced tumors undergo the distinctphases of tumor development and, importantly, allow the use ofimmunocompetent mice.

Prostate Cancer Model.

The TRAMPS mouse line is a widely used model of prostate cancer(Greenberg N M et al, 1995, Proc Natl Acad Sci USA. 92, 3439-3443). Theinitial lesions are prostatic intraepithelial hyperplasia (PIN) whichprogresses at about 12 weeks of age to a well differentiatedadenocarcinoma. Poorly differentiated adenocarcinomas arise in24-week-old TRAMP animals. At 18-24 weeks of age the TRAMP mice wereseparated into two groups, control and therapy, of 6 mice each.Anti-Jagged antibody 4D11 and IVIg control aliquots each were dosed IPto the respective group, q7DX5 at 20 mg/Kg. Seven days post final dosethe animals were sacrificed, and tumor burden was measured as weight ofgenitourinary tract and compared to control wild type C57/B16 mice. FIG.32 indicates that anti-Jagged antibody 4D11 was effective in limitingthe growth of Prostate tumors in TRAMP mice.

Breast Cancer Model.

A HER2/neu transgenic line develops mammary tumors in multiparousfemales at 20 weeks of age and 100% present lung metastasis at 25 weeksof age (Siegel P M et al., 1999, The EMBO Journal 18, 2149-2164). Forexperiments testing therapy for breast cancer, HER2/neu male mice werebred with FVB wild-type females and their progeny genotyped to selectfor HER2/neu females. At 20 weeks of age the HER2/neu females wereseparated into two groups, control and therapy. Anti-Jagged antibody4D11 and IVIg control aliquots each were dosed IP, q7DX5 at 20 mg/Kg.FIG. 33 indicates that anti-Jagged antibody 4D11 potently inhibited thegrowth of spontaneous tumors in Her2/neu transgenic mice.

Example 25 In situ Imaging of Labeled or Non-Labeled Anti-JaggedActivatable Antibodies with Detection by Secondary Antibodies

The present Example describes the use of in situ imaging of labeled ornon-labeled anti-Jagged activatable antibodies, wherein the cleavage andbinding were detected using a secondary antibody that specifically bindsto the AB portion of the activatable antibody. The results indicate theability to evaluate the activation and binding of non-labeledactivatable antibodies.

In situ imaging of the activation and binding of an Alexa680-labeledanti-Jagged activatable antibody 5342-1204-4D11 on TRAMP prostate cancertumor tissue was conducted as follows: Frozen tissue sections were laidover glass slides. A solution containing Alexa680-labeled anti-Jaggedactivatable antibodies was applied on the tissue and incubated, e.g.,for 1 hour at room temperature (about 22-24° C.) in an incubation bufferof 50 mM Tris-HCl buffer pH 7.4, containing 150 mM NaCl, 100 μM ZnCl₂, 5mM CaCl₂ and 0.05% Tween 20; activatable antibody at a concentration ofabout 4 μg/ml. The conditions of such an incubation can be adjusted tobe conducive to the cleavage agent in the tissue section by, forexample, varying the pH of the solution (e.g., within a range of aboutpH 7 to about pH 8.5), the temperature of the incubation (e.g., within arange of about 20° C. to about 40° C., e.g., room temperature or 37°C.), the incubation time (e.g., within a range of about 15 minutes toabout 150 minutes, and/or the activatable antibody concentrations (e.g.,within a range of about 0.05 μg/ml to about 10 μg/ml). The tissue wasthen extensively washed to remove non-bound material. The presence ofactivated antibody on the tissue was detected using imaging at 680 nmand a secondary anti-human IgG antibody labeled with AlexaFluor 488. Theconditions of that detection can be adjusted to the detecting reagentand detection modality (e.g., fluorescently labeled). For example, whena fluorescent tag was used, the tissue was submitted to fluorescentmicroscopy. As shown in FIG. 34, anti-Jagged activatable antibody5342-1204-4D11 demonstrated identical staining at both Alexa 680 (labelof anti-Jagged activatable antibody) and FITC channels (label ofanti-human IgG antibody), indicating that it is possible to conduct insitu imaging with non-labeled activatable antibodies and a secondaryreagent that specifically binds to the activatable antibody, such as alabeled antibody. The fluorescent signal shown in both channels wasinhibited by pre-treatment of the tissue with a 1:100 dilution of broadspectrum inhibitor cocktail set III (BSPI) (539134, EMD Millipore,Billerica, Mass.) and 50 μM of broad spectrum MMP inhibitor Galardin(Calbiochem, Millipore), as shown in FIG. 34, lower row.

Example 26 In Situ Imaging of Non-Labeled Anti-Jagged ActivatableAntibodies

The present Example describes the use of in situ imaging of non-labeledanti-Jagged activatable antibodies. The cleavage and binding weredetected using a secondary antibody that specifically binds to the ABportion of the activatable antibody.

In situ imaging of the activation and binding of a non-labeledanti-Jagged activatable antibody 5342-1204-4D11 on TRAMP prostate cancertumor tissue was conducted as follows: Frozen tissue sections were laidover glass slides. A solution containing non-labeled anti-Jaggedactivatable antibodies was applied on the tissue and incubated, e.g.,for 1 hour at room temperature (about 22-24° C.) in an incubation bufferof 50 mM Tris-HCl buffer pH 7.4, containing 150 mM NaCl, 100 μM ZnCl₂, 5mM CaCl₂ and 0.05% Tween 20; activatable antibody at a concentration ofabout 4 μg/ml. The conditions of such an incubation can be adjusted tobe conducive to the cleavage agent in the tissue section by, forexample, varying the pH of the solution (e.g., within a range of aboutpH 7 to about pH 8.5), the temperature of the incubation (e.g., within arange of about 20° C. to about 40° C., e.g., room temperature or 37°C.), the incubation time (e.g., within a range of about 15 minutes toabout 150 minutes, and/or the activatable antibody concentrations (e.g.,within a range of about 0.05 μg/ml to about 10 μg/ml). The tissue wasthen extensively washed to remove non-bound material. The presence ofactivated antibody on the tissue was detected using a secondaryanti-human IgG antibody labeled with AlexaFluor 488. The conditions ofthat detection can be adjusted to the detecting reagent and detectionmodality (e.g., fluorescently labeled). For example, when a fluorescenttag was used, the tissue was submitted to fluorescent microscopy. Asshown in FIG. 35, anti-Jagged activatable antibody 5342-1204-4D11demonstrated staining with comparable intensity and pattern as parentalanti-Jagged antibody (columns 2 and 1, respectively). The fluorescentsignal of anti-Jagged activatable antibody 5342-1204-4D11 was inhibitedby pre-treatment of the tissue with a 1:100 dilution of broad spectruminhibitor cocktail set III (BSPI) (539134, EMD Millipore, Billerica,Mass.) and 50 μM of broad spectrum MMP inhibitor Galardin (Calbiochem,Millipore), as shown in FIG. 35, column 3. The data demonstrate thefeasibility of conducting in situ imaging using non-labeled (i.e.,unlabeled) activatable antibodies and a secondary reagent that comprisesa detectable label and that specifically binds the AB of the activatableantibody.

Human triple-negative breast cancer (TNBC) and pancreatic cancer tissuesamples were profiled for the ability of anti-Jagged activatableantibody 5342-1204-4D11 to be activated and to bind human tumor using insitu imaging. The activatable antibody was labeled with Alexa Fluor® 680(Invitrogen) as described above. The resultant activatable antibody5342-1204-4D11-AF680 was incubated with frozen patient tissue samplesaccording to the protocol of in situ imaging described herein. Theresults on the ability of TNBC and pancreatic cancer patients' tissuesamples to activate and bind anti-Jagged activatable antibodies aresummarized in Table 27 and Table 28. The IHC staining that measures theamount of anti-Jagged (4D11) antibody binding to the tissue sample wasscored from − to 3+: −, no staining; 1+ (i.e., “+”), weak staining; 2+(i.e., “++”), moderate staining; and 3+ (i.e., “+++”), strong staining.The in situ imaging of anti-Jagged activatable antibodies staining wasquantified based on comparison with anti-Jagged antibody staining Table27 illustrates the expression level of Jagged 1 and/or Jagged 2 detectedby 4D11 binding and ability of triple-negative breast cancer (TNBC)tissues to activate and bind anti-EGFR activatable antibodies. Table 28illustrates the expression level of Jagged 1 and/or Jagged 2 detected by4D11 binding and the ability of pancreatic cancer tissues to activateand bind anti-EGFR activatable antibodies.

TABLE 27 Screening for Jagged 1 and/or Jagged 2 expression usinganti-Jagged antibody 4D11 and in situ imaging of anti-Jagged activatableantibody 5342-1204-4D11 in TNBC cancer patients' tumor tissues.5342-1204- Patient # Stage Her-2 neu ER PR 4D11 4D11 (%) 1 IIIA 0negative negative +++ 100 2 IIIB 0 negative negative + 5 3 IIA 0negative negative +++ 50 4 IIA 0 negative negative +++ 15 5 IIA 0negative negative +++ 85 6 IIIA 0 negative negative ++ 95 7 IIA  1+negative negative +++ 90 8 IIA 0 negative negative +++ 80 9 IIA  1+negative negative ++ 25 10 IIA 0 negative negative ++ 95

TABLE 28 Screening for Jagged 1 and/or Jagged 2 expression usinganti-Jagged antibody 4D11 and in situ imaging of anti-Jagged activatableantibody 5342-1204-4D11 in pancreatic cancer patients' tumor tissues.5342-1204- Patient # Diagnosis Stage Grade TNM status 4D11 4D11 (%)HF-0301-17 adenocarcinoma N/A G1 Well pT3, pN1b, + 35 Differentiated pMXHF-0301-19 adenocarcinoma N/A G2 Moderately pT4. pN1, +++ 45Differentiated pMX HF-0301-20 adenocarcinoma N/A G2 Moderately pT3,pN1b, + 55 Differentiated pMX HF-0301-21 adenocarcinoma N/A G3 PoorlypT3, pN1, ++ 100 Differentiated pMX HF-0301-22 adenocarcinoma N/A G2Moderately pT3, pN1, ++ 80 Differentiated pMX HF-0301-23 adenocarcinomaN/A Moderately to pT3, pN1, +++ 55 poorly pMX differentiated HF-0301-24adenocarcinoma N/A G2 to G3 pT3, pN1, +++ 100 Moderately to pM0 PoorlyDifferentiated HF-0301-10 adenocarcinoma IIB G1 Well PT3, pN1, ++ 100Differentiated pM n/a HF-0301-12 adenocarcinoma IIB G2 Moderately pT3,pN1, ++ 80 Differentiated pM n/a HF-0301-13 adenocarcinoma I G3 PoorlypT2, pN0, +++ 30 Differentiated pM n/a HF-0301-06 Ductal II G2Moderately pT3, pN1 ++ 100 Adenocarcinoma Differentiated HF-0301-16adenocarcinoma IIB G2 Moderately pT3, pN1 ++ 97 DifferentiatedHF-0301-15 adenocarcinoma IIB G2 Moderately pT3, pN1, ++ 50Differentiated pM n/a HF-0301-08 Ductal IIB G2 Moderately pT3, pN1 ++100 Adenocarcinoma Differentiated HF-0301-14 adenocarcinoma IB G3 PoorlypT2, pN0, − — Differentiated pM n/a TBD adenocarcinoma TBD TBD TBD ++ 80TBD adenocarcinoma TBD TBD TBD + 100 HF-0301-01 Ductal IIB G2 ModeratelypT2, pN1, ++ +++ Adenocarcinoma Differentiated pM n/a HF-0301-02adenocarcinoma II Moderately pT3, pN1, +++ ++ differentiated pMn/aHF-0301-03 adenocarcinoma II G2 Moderately pT3, pN0 ++ + DifferentiatedHF-0304-02 adenocarcinoma IIA G3 Poorly pT3, pN0, ++ + DifferentiatedpMn/a

Example 27 Protease Activation and Binding of an Anti-Jagged ActivatableAntibody

This Example demonstrates the ability of anti-Jagged antibody5342-1204-4D11 to be activated in vitro.

Anti-Jagged activatable antibody 5342-1204-4D11 was activated bycombining the activatable antibody and active site titrated MT-SP1 inPBS at final concentrations of 58.5 uM and 570 nM respectively. Themixture was incubated at 37° C. for 20 h. Prior to Protein Apurification, an aliquot was removed and analyzed by SDS-PAGE to confirmthat proteolytic digestion of 5342-1204-4D11 had gone to completion.

To remove the MT-SP1 and cleaved masking moiety, the activated5342-1204-4D11 was purified using standard Protein A chromatography.Briefly, a 1 mL Hi-Trap Protein A column (GE Healthcare life sciences)was equilibrated with PBS. The digested protein was bound to the columnand washed extensively with PBS. The bound protein was eluted using 1 MGlycine, pH 3.0 and neutralized with 0.1 M Tris, pH 8.0, andsubsequently dialyzed overnight into PBS.

The binding of anti-Jagged antibody 4D11, anti-Jagged activatableantibody 5342-1204-4D11, and anti-Jagged activated antibody5342-1204-4D11 to recombinant human Jagged 1-Fc was measured with anenzyme-linked immunosorbent assay (ELISA). Briefly, recombinant hJag1-Fc(R&D Systems) was absorbed to wells of a 96-well ELISA plate at aconcentration of 1 μg/ml in HANKS buffer overnight at 4° C. Allsubsequent steps were done at room temperature. The plates were blockedwith HANKS, 0.05% Tween, 4.0% non-fat dry milk for 1 hour. The 4D11antibody and activated antibody 5342-1204-4D11 were added to the plateat 100, 30, 10, 3, 1, 0.3, 0.1, and 0.03 nM and the activatable antibody5342-1204-4D11 was added to the plate at 1000, 300, 100, 30, 10, 3, 1,0.3 nM and incubated for 1 hour. All measurements were done intriplicate. After the plates were washed 5× with HANKS, 0.05% Tween ananti-human FAB-goat-HRP secondary (Sigma) was added to the plate at aconcentration of 1:5000 in HANKS, 0.05% Tween, 4.0% non-fat dry milk andincubated for 1 hour, washed 5× as before, and then developed using1-STEP-TMB1 ELISA solution (Thermo Scientific). The absorbance at 450 nmwas measured using a TECAN plate reader. FIG. 36 shows that the abilityof activated antibody 5342-1204-4D11 to bind Jagged 1 isindistinguishable from 4D11 antibody binding to Jagged 1.

Example 28 In Vivo Efficacy and Safety of an Anti-Jagged ActivatableAntibody in the H292 Tumor Model

Anti-Jagged activatable antibody 5342-1204-4D11 was tested for theability to reduce the growth of H292 xenograft tumors using thefollowing method. Female nu/nu mice, age 6-8 weeks, were implantedsubcutaneously with 5×10⁶ H292 cells in serum-free medium with matrigel(1:1). Tumors were measured every other day until 48-60 mice with tumorsin the target range (˜100-250 mm3) could be randomized into groups ofequal average tumor volume when the mean tumor size reached 150-200 mm3.(n=8-10/group). Animals were treated using the doses set forth in Table29.

TABLE 29 Groups and doses for the H292 efficacy study. Dose Dosing GroupN Treatment (mg/kg) Route Schedule 1 8 IVIg 20 mg/kg i.p q7dx 4 2 8 4D11(CTX-014)  2 mg/kg i.p. q7dx 4 3 8 4D11 (CTX-014) 6.7 mg/kg  i.p. q7dx 44 8 4D11 (CTX-014) 20 mg/kg i.p. q7dx 4 5 8 5342-1204-4D11 6.7 mg/kg i.p. q7dx 4 (CTX-033) 6 8 5342-1204-4D11 20 mg/kg i.p. q7dx 4 (CTX-033)

FIG. 37, which shows tumor volume, demonstrates that the anti-Jaggedactivatable antibody 5342-1204-4D11 inhibited the growth of the H292xenograft tumors. The serum concentration of mouse thymic stromallymphopoietin (TSLP) was measured as described above. The serum levelsof mouse TSLP (mTSLP) were quantified for individual mice uponsacrifice; results are shown in FIG. 38. Activatable antibody5342-1204-4D11 showed no elevation in TSLP while animals in the antibodyat 6.7 and 20 mg/kg showed increased TSLP as compared to the IVIgtreated group.

Upon sacrifice, skin was taken from the abdomen of animals treated with20 mg/kg of IVIg, antibody 4D11, or activatable antibody 5342-1204-4D11.The skin was formalin-fixed, paraffin-embedded and H&E stained. As FIG.39 depicts, hyperkeratosis was observed in the antibody-treated group,while the activatable antibody-treated group showed limited or nohyperkeratosis. The arrow points to a hair follicle showing significanthyperkeratosis.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. An isolated fully human monoclonal antibody thatbinds Jagged 1 and Jagged
 2. 2. The antibody of claim 1, wherein theantibody comprises a VH CDR1 sequence comprising the amino acid sequenceSYAMS (SEQ ID NO: 200); a VH CD2 sequence comprising the amino acidsequence SIDPEGRQTYYADSVKG (SEQ ID NO: 208); a VH CDR3 sequencecomprising the amino acid sequence DIGGRSAFDY (SEQ ID NO: 209); a VLCDR1 sequence comprising the amino acid sequence RASQSISSY (SEQ ID NO:210); a VL CDR2 sequence comprising the amino acid sequence AASSLQS (SEQID NO: 211); and a VL CDR3 sequence comprising the amino acid sequenceQQTVVAPPL (SEQ ID NO: 212).
 3. The antibody of claim 1, wherein theantibody comprises a combination of a VH CDR1 sequence, a VH CDR2sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2 sequence,and a VL CDR3 sequence selected from the combinations shown in Table 2.4. The antibody of claim 3, wherein the antibody comprises a combinationof a variable heavy chain region and a variable light chain region fromthe combinations listed in Table
 4. 5. The antibody of claim 1, whereinthe antibody binds Jagged 1 and Jagged 2 and prevents one or more ofJagged 1 or Jagged 2 from binding a Notch receptor.
 6. The antibody ofclaim 1, wherein said antibody is an IgG isotype.
 7. The antibody ofclaim 6, wherein said antibody is an IgG1 isotype.
 8. The antibody ofclaim 1 comprising an agent conjugated to the antibody.
 9. The antibodyof claim 8, wherein the agent is a therapeutic agent, an antineoplasticagent, a toxin or fragment thereof, a detectable moiety or a diagnosticagent.
 10. The antibody of claim 8, wherein the agent is conjugated tothe antibody via a linker.
 11. The antibody of claim 10, wherein thelinker is a cleavable linker.
 12. A pharmaceutical compositioncomprising an antibody of claim 1 and a carrier.
 13. An activatableantibody that in an activated state binds Jagged 1 and Jagged 2comprising: an antibody or an antigen binding fragment thereof (AB) thatspecifically binds to Jagged 1 and Jagged 2; a masking moiety (MM) thatinhibits the binding of the AB to Jagged 1 and Jagged 2 when theactivatable antibody is in an uncleaved state; and a cleavable moiety(CM) coupled to the AB, wherein the CM is a polypeptide that functionsas a substrate for a protease.
 14. The activatable antibody of claim 13,wherein the MM has an equilibrium dissociation constant for binding tothe AB which is greater than the equilibrium dissociation constant ofthe AB to Jagged 1 and Jagged
 2. 15. The activatable antibody of claim13, wherein the MM does not interfere or compete with the AB for bindingto Jagged 1 and Jagged 2 when the activatable antibody is in a cleavedstate.
 16. The activatable antibody of claim 13, wherein the protease isco-localized with Jagged 1 and/or Jagged 2 in a tissue, and wherein theprotease cleaves the CM in the activatable antibody when the activatableantibody is exposed to the protease.
 17. The activatable antibody ofclaim 13, wherein the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM.
 18. The activatable antibody of claim 13, whereinthe activatable antibody comprises a linking peptide between the MM andthe CM.
 19. The activatable antibody of claim 13, wherein theactivatable antibody comprises a linking peptide between the CM and theAB.
 20. The activatable antibody of claim 13, wherein the activatableantibody comprises a first linking peptide (LP1) and a second linkingpeptide (LP2), and wherein the activatable antibody follows in theuncleaved state has the structural arrangement from N-terminus toC-terminus as: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
 21. The activatableantibody of claim 20, wherein the two linking peptides need not beidentical to each other.
 22. The activatable antibody of claim 20,wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids inlength.
 23. The activatable antibody of claim 13, wherein the MM is apolypeptide of about 2 to 40 amino acids in length.
 24. The activatableantibody of claim 13, wherein the MM polypeptide sequence is differentfrom that of Jagged 1 and Jagged 2 and wherein the MM polypeptidesequence is no more than 50% identical to any natural binding partner ofthe AB.
 25. The activatable antibody of claim 13, wherein the CM is apolypeptide of up to 15 amino acids in length.
 26. The activatableantibody of claim 13, wherein the antigen binding fragment thereof isselected from the group consisting of a Fab fragment, a F(ab′)₂fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody,and a single domain light chain antibody.
 27. The activatable antibodyof claim 13, wherein the CM is a substrate for an enzyme selected fromthe group consisting of uPA, legumain, MT-SP1, ADAM17, BMP-1, TMPRSS3,TMPRSS4, MMP-9, MMP-12, MMP-13, and MMP-14.
 28. The activatable antibodyof claim 13 comprising an agent conjugated to the AB.
 29. Theactivatable antibody of claim 28, wherein the agent is a therapeuticagent, an antineoplastic agent, a toxin or fragment thereof, adetectable moiety or a diagnostic agent.
 30. The activatable antibody ofclaim 28, wherein the agent is conjugated to the AB via a linker. 31.The activatable antibody of claim 30, wherein the linker is a cleavablelinker.
 32. The activatable antibody of claim 13, wherein the MMcomprises a sequence selected from the group consisting of the sequencesshown in Table 9, Table 11, Table 12, Table 13, Table 14, Table 19,Table 20, Table 21, Table 22, or Table
 23. 33. The activatable antibodyof claim 13, wherein the antibody or antigen-binding fragment thereofthat binds Jagged 1 and Jagged 2 comprises a VH CDR1 sequence comprisingthe amino acid sequence SYAMS (SEQ ID NO: 200); a VH CD2 sequencecomprising the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO: 208); aVH CDR3 sequence comprising the amino acid sequence DIGGRSAFDY (SEQ IDNO: 209); a VL CDR1 sequence comprising the amino acid sequenceRASQSISSY (SEQ ID NO: 210); a VL CDR2 sequence comprising the amino acidsequence AASSLQS (SEQ ID NO: 211); and a VL CDR3 sequence comprising theamino acid sequence QQTVVAPPL (SEQ ID NO: 212).
 34. The activatableantibody of claim 13, wherein the antibody or antigen-binding fragmentthereof that binds Jagged 1 and Jagged 2 comprises a combination of a VHCDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1sequence, a VL CDR2 sequence, and a VL CDR3 sequence selected from thecombinations shown in Table
 2. 35. The activatable antibody of claim 13,wherein the antibody or antigen-binding fragment thereof that bindsJagged 1 and Jagged 2 comprises a combination of a variable heavy chainregion and a variable light chain region from the combinations listed inTable
 4. 36. The activatable antibody of claim 13 comprising a lightchain amino acid sequence selected from the group consisting of SEQ IDNOs: 74, 132, 134, 136, 138, 140, 142, 144, 146, 180, 182, 184, 186,188, 190, 192, 194, and 196, and a heavy chain amino acid sequenceselected from the group consisting of SEQ ID NOs: 76 and
 148. 37. Anisolated nucleic acid molecule encoding the antibody of claim
 1. 38. Avector comprising the isolated nucleic acid molecule of claim
 37. 39. Anisolated nucleic acid molecule encoding the activatable antibody ofclaim
 13. 40. A vector comprising the isolated nucleic acid molecule ofclaim
 39. 41. A method of producing an antibody or an activatableantibody by culturing a cell under conditions that lead to expression ofthe antibody or the activatable antibody, wherein the cell comprises thenucleic acid molecule of claim
 37. 42. A method of producing an antibodyor an activatable antibody by culturing a cell under conditions thatlead to expression of the antibody or the activatable antibody, whereinthe cell comprises the nucleic acid molecule of claim
 39. 43. A methodof manufacturing an activatable antibody that binds Jagged 1 and Jagged2 in an activated state, the method comprising: (a) culturing a cellcomprising a nucleic acid construct that encodes the activatableantibody under conditions that lead to expression of the activatableantibody, wherein the activatable antibody comprises a masking moiety(MM), a cleavable moiety (CM), and an antibody or an antigen bindingfragment thereof (AB) that specifically binds Jagged 1 and Jagged 2, and(b) recovering the activatable antibody.
 44. The method of claim 43,wherein the CM is a polypeptide that functions as a substrate for aprotease.
 45. The method of claim 43, wherein the CM is positioned inthe activatable antibody such that, in an uncleaved state, the MMinterferes with specific binding of the AB to Jagged 1 and Jagged 2 andin a cleaved state the MM does not interfere or compete with specificbinding of the AB to Jagged 1 and Jagged 2
 46. A method of alleviating asymptom of a clinical indication associated with cancer in a subject,the method comprising administering the antibody of claim 1 to a subjectin need thereof in an amount sufficient to alleviate the symptom of theclinical indication associated with cancer.
 47. A method of alleviatinga symptom of a clinical indication associated with cancer in a subject,the method comprising administering the activatable antibody of claim 13to a subject in need thereof in an amount sufficient to alleviate thesymptom of the clinical indication associated with cancer.
 48. A methodof reducing angiogenesis comprising administering the antibody of claim1 to a subject in need thereof in an amount sufficient to reduceangiogenesis.
 49. A method of reducing angiogenesis comprisingadministering the activatable antibody of claim 13 to a subject in needthereof in an amount sufficient to reduce angiogenesis.
 50. A method ofreducing Jagged 1 and/or Jagged 2 signaling comprising administering theantibody of claim 1 to a subject in need thereof in an amount sufficientto reduce Jagged 1 and/or Jagged 2 signaling.
 51. A method of reducingJagged 1 and/or Jagged 2 signaling comprising administering theactivatable antibody of claim 13 to a subject in need thereof in anamount sufficient to reduce Jagged 1 and/or Jagged 2 signaling.
 52. Themethod of claim 46, wherein said subject is a human.
 53. A method ofalleviating a symptom of a fibrotic disorder, wherein the methodcomprises administering the antibody of claim 1 to a subject in needthereof in an amount sufficient to alleviate the symptom of the fibroticdisorder in the subject.
 54. A method of alleviating a symptom of afibrotic disorder, wherein the method comprises administering theactivatable antibody of claim 13 to a subject in need thereof in anamount sufficient to alleviate the symptom of the fibrotic disorder inthe subject.
 55. The method of claim 53, wherein said subject is ahuman.